J. Biochem., 81, 555-562 (1977)

Fine Structure of SMG Alginate Fragment in the Light of Its Degradation by Alginate Lyases of Pseudomonas sp. Kyung Hee MIN, 1 Sonoko F. SASAKI, 1 Yoshiko KASHIWABARA,' Makoto U M E K A W A / and Kazutosi NISIZAWA' Department of Botany, Faculty of Science, Tokyo Kyoiku University, Otsuka, Bunkyo-ku, Tokyo Received for publication, May 12, 1976

An alginate fragment named SMG, consisting of mannuronic (M) and guluronic acid residues (G) (DP=25), was prepared from the partial acid hydrolysate of a commercial alginate. Two subfractions, SMG-ppt (DP=52) and SMG-sup (DP = 18) were obtained from SMG by fractionation with MgCl 2 and CaG 2 . The M/G ratios of these alginate fragment were 1.4-1.9. Their lysis products by a pseudomonad alginate lyase [EC 4.2.2.3] preparation were fractionated by gel filtration, giving similar patterns. The major products in their digests were unsaturated monouronides (53-50%) and triuronides (30-35 %). The former was identified as a J4,5-hexuronic acid (^JU) and the latter was identified as a mixture of J4,5-hexuronosyl-(l—> 4)-/}-D-mannuronosyl-(l—»4)-L-guluronic acid (4UMG) and J4,5-hexuronosyl-(l—*4)-a-Lguluronosyl-(l—>4)-L-guluronic acid (JUGG). The two unsaturated triuronides were present in roughly equal amounts. The presence of 4-O-a-L-guluronosyl-L-guIuronic acid (GG) and 4-O-/3-D-mannuronosyl-L-guluronic acid (MG) or 4-0-)9-L-guluronosyl-D-mannuronic acid (GM) was also demonstrated in the digest. Moreover, indirect evidence suggested nonreducing terminal A\J residue and free J U in the digest to be derived more from M than G of the original SMG. Thus, it was concluded that more than one-third of uronic acid residues of SMG molecules may be composed of almost equal amounts of MG and GG sequences, most of which may be connected with M to form MMG and MGG sequences, respectively. 1

Present address: Department of Biology, College of Liberal Arts and Sciences, Suk-Mung Women's University, Seoul, Korea. • Present address: Department of Home Science, Faculty of Humanity and Culture, Tokai University, Hiratsuka, Kanagawa 254. 1 Present address: National Institute for Leprosy Research, Higashimurayama, Tokyo 189. 4 Present address: Knorr Foods Co., Takatsu-ku, Kawasaki, Kanagawa 210. * Present address: Department of Fisheries, College of Agriculture and Veterinary Medicine, Nihon University, Setagaya-ku, Tokyo 154. Abbreviations- M, mannuronic acid residue; G, guluronic acid residue; MA, D-mannuronic acid; GA, L-guluronic acid; SM, alginate fragment of polymannuronide; SG, alginate fragment of polyguluronide; SMG, alginate fragment of polyuronide consisting of M and G; GG, 4-O-a-L-guluronosyl-L-guluronic acid; GM, 4-O-/3-L-guluronosyl-Dmannuronic acid; JU, J4,5-hexuronicacid; JUMM, J4,5-hexuronosyl-{l—»4)-/3-D-mannuronosyl-(l—>4)-D-mannuronic acid; JUGG, J4,5-hexuronosyl-(l—>4>/3-L-guluronosyl-(l—>4>L-guluromc acid; JUMG, J4,5-hexuronosyl(1—>4)-/3-D-mannuronosyl-{l—>4)-L-guluronic acid; SMG-sup, SMG subfraction in the supernatant of CaCI,-treated SMG solution; SMG-ppt, SMG subfraction precipitated from CaCl,-treated SMG solution; GLC, gas-liquid chromatography; TMS, trimethylsilylation; DP, average degree of polymerization.

Vol. 81, No. 3, 1977

555

556

K.H. MIN, S.F. SASAKI, Y. KASHIWABARA, M. UMEKAWA, and K. NISIZAWA

It has been established that alginate molecules contain two kinds of uronic acid residues, M and G (1, 2), each of which occurs not only in individual blocks but also in mixed blocks (3-6). The chain lengths of these blocks are not known at present, but hetero-polyuronides of DP 15-40 (4, 7) containing both residues and polymannuronides and -guluronides of DP 10-28 (3,5,7) have been obtained from partial acid hydrolysates of alginates. The structure of the hetero-alginate fragments, particularly the sequence of M and G, however, remains obscure, except for some information reported by Haug et al. (4). By identification of the diuronides derived from a hetero-polyuronide fragment (named Preparation A, DP=20), these authors assumed a structure consisting of alternating M and G sequences. This structure was supported by Larsen et al. (8). They calculated the amounts of oligouronides in the partial acid hydrolysate of an alginate fragment (named fragment A, DP=19) on the basis of the Kuhn equation (9, 10), and compared the results with those obtained for polymannuronide and polyguluronide. Thus, they arrived at the conclusion that 90% of fragment A was of alternating structure, and the average length of the alternating parts was estimated by them to be at least 10 residues. Using alginate lyases, on the other hand, Nisizawa et al. (7) also studied the fine structure of hetero-alginate fragments which they designated as SMG and seemed to correspond to Larsen's fragment A. SMG from Ishige okamurai alginates has been reported to be degraded by polyguluronide lyase from a pseudomonad (11) as easily as SG, while it was hardly attacked by polymannuronide lyase from a mollusc, Dolabella auricula (5) which readily degraded SM. Therefore we attempted to elucidate the sequence of M and G of SMG using the pseudomonad polyguluronide lyases, since the lyases produced a mixture of JUGG and JUGM or 4UMG from SMG in almost equal amounts, together with J U (12). In this paper the results are reported. MATERIALS AND METHODS Enzyme Source—An alginate lyase [EC 4.2.2.3] preparation was obtained by extraction from

sonicated cells of Pseudomonas sp. No. 9 cultured on alginate medium (//). This preparation contained at least three endo-polyguluronide lyases, but was practically free of polymannuronide lyase. Moreover, all three lyase components degraded SMG and produced a mixture of unsaturated uronides (12). Therefore, the preparation was used without further purification to obtain unsaturated oligouronides from SMG. Preparation of SMG—SMG alginate fragment was prepared by the method of Haug et al. (3). A commercial alginate (50 g) was hydrolyzed with 500 ml of 1 M oxalic acid at 100°C for 20 min. This treatment was repeated once. The soluble materials obtained by filtration were combined, neutralized with sodium carbonate solution and concentrated under reduced pressure. The concentrate was dialyzed against running tap water. The dialyzed solution was further concentrated under reduced pressure to 100-150 ml and ethanol was added. The resulting precipitate was centrifuged at 1,000 x g for 20 min, washed with ethanol and acetone, and dried in a desiccator. The DP estimated by reducing end assay with the chromotropic acid reagent was 25 (75), and the M/G ratio was 1.4-1.9, depending on the starting alginate lot. Yields were 2.4-3.4 g. Preparation of SMG-ppt and SMG-sup from SMG—SMG (M/G ratio = 1.4) was fractionated further with MgCl, and CaCl, by the method of Haug et al. (4) to obtain subfractions. To a concentrated solution of SMG (7.5g/1.5 litters) obtained from 200 g of an alginate preparation, a concentrated MgClt solution was added to a final concentration of 0.2 M, then half a volume of 0.1 M CaG, was added. The precipitate (crude SMGppt) was collected by centrifugation, dissolved in 0.6 M EDTA, and dialyzed against distilled water. The supernatant (crude SMG-sup) was treated with 0.6 M EDTA after dialysis and then dialyzed again in the same way. The two crude subfractions were finally lyophilized. From 600 g of the alginate, 14 g of crude SMG-ppt and 18 g of crude SMG-sup were obtained. Their DP's were estimated by means of the chromotropic acid reagent (13) to be 43 and 16, respectively, but their M/G ratios were approximately the same as that of the starting SMG. Next, the crude SMG-ppt and SMG-sup subfractions were each (1.5 g) dissolved in 20 ml of water and fractionated by gel nitration on Sephadex J. Biochem.

FINE STRUCTURE OF SMG ALGINATE FRAGMENT G-75 and G-25 columns (4x145 cm and 3x175 cm), respectively, using 0.1 N NaCl as an eluent. The eluates were collected in fractions of 20 ml and uronides were detected by the phenol-sulfuric acid method (14). The SMG-ppt showed a wide molecular weight distribution, with no particular peak, over fractions, 30 to 80, while SMG-sup was separated into two peaks, one from fractions 20 to 36 and the other from fractions 37 to 62. Major fractions (No's. 34-64, ca. 81 % of the total) from SMG-ppt and the first peak (No's. 20-36, ca. 40% of the total) from SMG-sup were individually pooled, desalted on Sephadex G-15 and lyophihzed. Purification on a similar scale was carried out repeatedly for the remaining crude samples. The total recoveries of SMG-ppt and -sup were 3.0 g and 2.0 g, respectively, and the DP's of purified SMG-ppt and SMG-sup were estimated by the chromotropic acid method (75) to be 52 and 18, respectively. Their M/G ratios remained almost unchanged. Identification of Reducing End Residues of Unsaturated Tri- tand Di-uronides by GLC—A stainless-steel GLC column (0.3 cm x 1 m) was packed with 10% (w/w) neopentyl-glycol sebacate polyester on 80-100 mesh acid-washed Chromosorb W (15), and connected to a Hitachi 063 gas chromatograph. Aldonolactones obtained from the reducing terminals of unsaturated uronides after reduction with NaBH4 and acid hydrolysis were trimethylsilylated. The O-trimethylsilylaldonolactones thus obtained were dissolved in pyridine and injected into the column with a microsyringe (1 (A), and developed at 170°C with nitrogen gas at a flow rate of 45 ml per min. Separation of Monouronic Acids on an Anion Exchange Column—Uronic acid components of SMG and unsaturated oligouronides obtained from lysis products were separated by chromatography on an anion exchange column packed with Dowex 1 x8, (acetate form) 200-400 mesh (1.1 x 14 cm), as follows (16). The samples were hydrolyzed with an equal volume of 2 N sulfuric acid for 6 h, neutralized with CaCOj, desalted and concentrated by evaporation under reduced pressure. The concentrate was applied to the Dowex column. Elution was carried out with a linear gradient of acetic acid at increasing concentration from 0.5 (200 ml) to 2 N (200 ml) at a flow rate of 0.3-0.5 ml per min. The eluates were collected in Vol. 81, No. 3, 1977

557

fractions of 4 ml and the fractions positive to phenol-sulfuric acid reagent (14) were further examined by paper chromatography. Analytical Methods—Total uronic acid was determined by the phenol-sulfuric acid method (14). 4, 5-Unsaturated uronic acid (4U) was determined by the TBA reagent method (17). The reducing power of uronides was determined by the method of Somogyi (18). Paper Chromatography of Uronic Acid—Paper chromatography for the identification of enzymic degradation products was carried out on Toyo No. 50 and Whatman 3 MM filter papers by the descending technique, and spots were detected by treatment with silver nitrate (black coloring) and thiobarbituric acid reagents (red coloring), as described in a previous paper (12). A modified solvent of Yoshikawa et al. consisting of 1-butanol: methanol: water (5 : 3 : 1, v/v) (Solvent A) was used for the separation of saturated and unsaturated monomers (19) and pyridine : ethyl acetate : acetic acid : water ( 5 : 5 : 1 : 3 , v/v) (Solvent B) for the separation of di- and triuronides, saturating the chromatographic chamber with pyridine : ethylacetate : water (11 : 40 : 6, v/v) (1). RESULTS Isolation of the Products Formed from SMG by Alginate Lyase—SMG (500 mg) was digested with alginate lyase (100 mg) in 0.01 M Tris-HCl buffer (500 ml), pH 8.0, containing 0.2 N NaCl at 30°C for 3 days. The reaction mixture was boiled for about 5 min, and divided into 10 portions. Each one was fractionated by gel filtration on a Sephadex G-25 column (3 x 175 cm), using 0.1 N NaCl as an eluent, and fractions of 10 ml were collected (Fig. 1). Fractions in the tnmer region (No's. 74 to 84), dimer region (No's. 85 to 92), and monomer region (No's. 93 to 104) from each gel filtration were pooled separately, desalted on a Sephadex G-15 column using distilled water as an eluent, and concentrated by evaporation. Yields on the basis of TBA reaction for these products were 34.7 % for trimer, 11.5% for dimer, and 53.4% for monomer, indicating that the initial SMG used as a substrate had been almost completely degraded into these products. Identification of Products in the Monomer Region—The products in the monomer region (1.7

K.H. MIN, S.F. SASAKI, Y. KASHIWABARA, M. UMEKAWA, and K. N1SIZAWA

558

observed with solvent B, whose identification was not attempted in the present work. Next, the unsaturated monomer was characterized by periodate oxidation after reduction {13). An aqueous solution of the sample (21 mg) was reduced with 0.1 N sodium borohydride in 20 ml of 0.5% boric acid at pH 7.0 and allowed to stand overnight at 5°C. Sodium ions were removed with Amberlite LR 120 (H + ) and boric acid was evaporated off as methyl borate. The dried residue was dissolved in water (1 ml) and oxidized in a cold room with 0.015 M sodium periodate in 0.05 M acetate buffer, pH 4.3, in a final volume of 20 ml. When the periodate consumption had been ceased, formaldehyde produced was determined by the chromotropic acid method. 50

60

70

80

90

100

FRACTION NUMBER

Fig. 1. Gel filtration of lysis products formed from SMG by alginate lyase on a Sephadex G-25 column. • , amounts of products as determined by the phenolsulfunc acid method, using authentic guluronic acid as a standard; O, thiobarbituric acid method, using authentic unsaturated mono-, di-, and triuronides as standards. Fraction volume, 10 ml per tube; void volume, around 600 ml.

per ml) were applied to a carbon column ( 4 x 6 cm), and eluted stepwise with water and 20% ethanol. The first eluate with water yielded only a little material, which was positive to silver nitrate reagent, but negative in the TBA test. The product was, however, not identified because of the small amount available. The eluates with 20% ethanol were TBA-positive. They were concentrated, dried and redissolved in water (11.2 mg per 16 ml). The yield was almost 80% of the initial material. This compound gave a single spot on the paper chromatogram with solvent A, but another faint tailing spot positive in the TBA test was often

On the other hand, another aliquot of unsaturated monomer was subjected to the TBA test to check for the presence of an A A, 5 bond in the pyranose ring (Table I). The reduced JU, which should give no formylpyruvate in the TBA test produced 0.46 /imol of formylpyruvate in this experiment. It is not clear at present whether this formylpyruvate is real or an artifact. However, it may be said on the basis of these experiments that the monomer tested, or at least most of it, is a 4, 5unsaturated hexuronide (d\J). Identification of Products in the Trimer Region —Uronides in the trimer region were separated by paper chromatography into four compounds. Two of them seemed to be trimers and the other two were apparently contaminating dimers. They were disignated dimers I, II and trimers I, II in order of their mobility on a paper chromatogram. Thus, 21 mg of trimer I, 19.5 mg of trimer II, 8.1 mg of dimer I, and 7.4 mg of dimer II were obtained by preparative paper chromatography. Analytical data for these uronides are summarized in Table II. Based on these results, dimer II and trimer II appear to be either GM or MG and JUGM or JUMG, respectively.

TABLE I. Chemical characterization of unsaturated monouronic acid by periodate oxidation. NaIO4 consumption (/imol) Before reduction After reduction Theoretical values:

a

6.2 /imol,

HCHO released (/imol)

—

—

6.6*

3.3b

b

Formyl pyruvate released (/imol) 3.05" 0.46

3.1 /imol.

/ . Biochem.

559

FINE STRUCTURE OF SMG ALGINATE FRAGMENT TABLE II. Some physical and chemical properties of dimers I, II and trimers I, II. Uronide sample

Mobility on paper chromatography

TBA reaction

RDi.I

Dimer I Dimer II

0.46 (equal to JUGG) Trimer II 0.25 Trimer I

Uronic acid after hydrolysis with 1 N H.SO, at 100°C for 5 h

Reducing power after hydrolysis (times)

Postulated structure

0

GA

ca. 2

GG

-

0

MA' GA*

ca. 2

GM or MG

,

ca. 1/3

GA

ca. 2

AUGG

+

ca. 1/3

MA"

ca. 2

JUGM or JUMG

1.0

0.79

TBA-absorbance ratio of equal amounts of sample and AU

» and b : The amounts of MA and GA were estimated by paper chromatography to be practically equal.

The sugar residue sequences of these uronides were further examined by GLC by Perry and Hulyalkar (15), as follows. Trimer II (6.5 mg as uronic acid/ml) and dimer II (4 mg as uronic acid/ml) solutions were each reduced with sodium borohydride (60 mg) in distilled water (15 ml). The reduced products were dissolved in water (1 ml), and hydrolyzed with 1 N HISO 4 at 100°C for 5 h. It was found by paper chromatography that both free and bound JU's are decomposed by this acid hydrolysis. After neutralization with CaCO3, the solution was desalted on a Sephadex G-10 column. The eluates positive in the phenolsulfuric acid reaction were pooled, concentrated and dried under reduced pressure. The dried hydrolysate was converted to 1,4lactones by evaporation with dilute HC1 then TMS reagent (0.1 ml) was added with shaking. As controls, both reduced and nonreduced mannuronic and guluronic acids, and reduced hydrolysates of both the unsaturated trimannuronide (JUMM) and SMG were lactonized and tnmethylsilylated in the same way. As shown in Fig. 2 the TMS derivatives of reduced guluronic (D-gulonic) and mannuronic (Lmannonic) acids gave peaks with retention times of 6 and 12 min, respectively. The TMS derivative of nonreduced guluronic as well as mannuronic acid gave no peak under the present conditions, even when large amounts were applied. Therefore, the TMS compounds from dimer II and trimer II corresponding to TMS-D-gulonic acid should be derived from the reducing ends of these uronides, and the structures of these uronides must be MG Vol. 81, No. 3, 1977

Guluronic sad —

Reduction TMS

Mannulontc acid —

Reduction TMS

Hydrolysis Reduction TMS

SMG

Hydrolysis Reduction TMS

Dimer II

Reduction Hydrolysis TMS Reduction

Trimer II

Hydrolysis

TMS

0

6

12

TIME (mm)

Fig. 2. GLC of reduced uronic acids from reduced mono-, oligo-, and polyuronides.

and AUMG, respectively. These findings also suggest that the endo-polyguluronide lyases used for the digestion of SMG split this alginate fragment at G-G and/or G-M bonds, as would be expected from the substrate specificity of these lyases. Original Residue of Free and Bound AU's Produced from SMG—SMG (15.5 mg) with an M/

K.H. MIN, S.F. SASAKI, Y. KASHIWABARA, M. UMEKAWA, and K. NISIZAWA

560

G ratio of 1.9 was digested at pH 8.0 with 20 ml of crude enzyme (2.5 mg, 110 units (12)) at 30°C. After incubation of two (B and C) reaction mixtures for 5 h and 3 days, respectively, each mixture was heated at 100°C for 5 min, concentrated to 5 ml and hydrolyzed with 1 N HtSO4 at 100°C for 6h.

20

30

40

FRACTION NUMBER

Fig. 3. Changes of uronic acid contents in the lytic •digests of SMG after incubation for 5 h (B) and 3 days u"> rragm Fig. 4. Paper chromatograms of trimers produced by curred at the bond between the 6th carbon atom alginate lyase from SMG-ppt, SMG-sup, and Frag- (the carbon of the carboxy group) and the 5th carment A. bon atom of the unsaturated monouronide (dU). In the present work, it was found that the to 53.4% and from 29.7 to 35.1 %, for monouronide amounts of M and G in SMG used as a substrate and triuronide, respectively. Paper Chromatographic Comparison of Two decreased with incubation time during degradation Kinds of Trimers from SMG-ppt, SMG-sup, and by an alginate lyase preparation, but the deFragment A—The trimer fractions eluted from lysis crease in M was much faster than that of G. This products of SMG-ppt, SMG-sup, and fragment A indicates that M may be converted into free and were applied, after desalting, to Whatman 3MM bound /JU's faster than G, since free and bound paper, and developed in solvent B for about 2 weeks. JU's were decomposed by acid hydrolysis used for The spots were detected with silver nitrate reagent the determination of M and G of SMG and only the remaining intact M and G were measurable. (Fig. 4).

II

Vol. 81, No. 3, 1977

562

K.H. MIN, S.F. SASAKI, Y. KASHIWABARA, M. UMEKAWA, and K. NISIZAWA

On the other hand, trimers from SMG consist of almost equal amounts of 4UGG and JUMG, and they amounted to 30-35% of the original SMG. Similar results were obtained with the subfractions, SMG-ppt and -sup (Table IV). Moreover, the presence of MG and GG was demonstrated in the dimers; GG and MG sequences may amount to more than 30-35% of the total residues of SMG. As discussed above, if the A\J residues of unsaturated oligouronides including JUGG and JUMG originate more from M than from G, it is possible that the sequences of MGG and MMG may be present more in the SMG than those of GGG and GMG. The amount of free 4U was also very large, more than 50% in SMG. The reason for this greater production of d\J is unclear at present, because no detailed examination has been carried out of the mode of action of endo-polyguluronide lyase from our pseudomonads on oligouronides. Possibly, however, A\J may be produced at the nonreducing end of intermediary oligouronides larger than tetramer by randomwise or endowise splitting by the endo-alginate lyases, since these enzymes showed no activity toward unsaturated t rimer. Results similar to those obtained with SMG and its subfractions were also obtained with fragment A, except that the content of AXJGG was roughly two-thirds of that of JUMG, and the production of A\J was relatively small. However, the sum of JUGG plus JUMG amounted to more than 30%, and this indicates that at least one-third of fragment A consists of sequences including MGG and MMG. Therefore, if this fragment A contains alternating sequences of M and G, as has been reported by Larsen et al. (8), such a structure may account for at most two-thirds of the total. Thus, polyuronide blocks consisting of M and G in alginic acid appear to be very complex as regards the sequence of their sugar residues. Note added in proof: After contribution of the manuscript of this work, we found that the results partly similar to our conclusion for the fine structure of fragment A has been reported by Simionescu et al. on the basis of analytical approach different from ours (20).

The authors are greatly indebted to Dr. B. Larsen, Institute for Marine Biochemistry, Trondheim, for providing the alginate fragment used in this work. REFERENCES 1. Fischer, F.G. & Dorfel, H. (1955) Z. physiol. Chem. 301, 224-234 2. Fischer, F.G. & Ddrfel, H. (1955) Z. physiol. Chem. 302, 186-203 3. Haug, A., Larsen, B., & Smidsrod, O. (1966) Ada Chem. Scand. 20, 183-190 4. Haug, A., Larsen, B., & Smidsred, O. (1967) Ada Chem. Scand. 21, 691-704 5. Nisizawa, K , Fujibayashi, S , & Kashiwabara, Y. (1968) / . Biochem. 64, 24-37 6. Fujibayashi, S., Habe, H., & Nisizawa, K. (1970) / . Biochem. 67, 37-45 7. Nisizawa, K., Sasaki, F.S., & Saigo, M. (1972) Proc. VII Intern. Seaweed Symp. (Nisizawa, K. et al., eds.) pp. 485-490, Univ. Tokyo Press, Tokyo 8. Larsen, B., Smidsred, O., Haug, A., & Painter, T. (1969) Ada Chem. Scand. 23, 2375-2388 9. Freudenberg, K., Kuhn, W., Durr, W., Holz, F., & Steinbrunn, G. (1930) Ber. B. 63, 1510-1530 10 Kuhn, W. (1930) Ber. B 63, 1503-1509 11. Kashiwabara, Y., Suzuki, H., & Nisizawa, K. (1969) / . Biochem. 66, 503-512 12. Min, K.H., Sasakj, F.S , Kashiwabara, Y., Suzuki, H., & Nisizawa, K. (1977) / . Biochem. 81, 539-546 13. John, C.S. (1962) in Methods in Carbohydrate Chemistry (Whistler, R.L., Wolfrom, M.L., BeMiller, J.N., & Shafizadeh, F., eds.) Vol. 1, pp. 441-445, Academic Press, New York and London 14. Dubois, M , Gilles, K., Hamilton, J.K., Rebers, P.A., & Smith, F. (1956) Anal. Chem. 28, 350-356 15. Perry, M.B. & Hulyalkar, R.K. (1965) Can. J. Biochem. 43, 573-584 16. Larsen, B. & Haug, A. (1961) Ada Chem. Scand. 15, 1397-1398 17. Weissbach, A. & Hurwitz, J. (1959) / . Biol. Chem. 234, 705-709 18. Somogyi, M. (1952) / . Biol. Chem. 195, 19-23 19. Yoshikawa, M. & Kiyohara, T. (1964) Sci. Rep. Hyogo Univ. Agr. Series • Agr. Chem. 6, 51-56 20. Siminonescu, CR.I., Popa, V.I., Ru5an, V., & Liga, A. (1975) Cellulose Chem. Technol. 9, 213-225

/ . Biochem.