Microbial polysaccharides containing 6-deoxysugars

1::3

Microbial polysaccharides containing 6-deoxysugars M A R I A N N E GRABER, ANDRI~ MORIN,* FRANCIS D U C H I R O N and PIERRE F. MONSAN BioEurope, 4, impasse Didier Daurat, Z.I. Montaudran, 31400 Toulouse, France

Summary. Microorganisms producing polysaccharides rich in 6-deoxysugars are widely distributed among bacteria, algae and fungi isolated from different environments and having different metabolic capabilities. Culture conditions leading to the screening of microorganisms producing these polysaccharides as well as parameters promoting their production necessitated the use of media containing high carbon-nitrogen ratios. Methods for extracting polysaccharides and for measuring their 6-deoxysugars content are either colorimetric or required the use of analytical chromatography. Successful purification procedures leading to pure 6-deoxysugars included ion-exchange chromatography. Among the different applications of these polysaccharides is their use as substrates in the chemical synthesis of flavoring agents.

Keywords: Microbial polysaccharides; furaneol; flavoring agent; 6-deoxysugars;rhamnose;fucose

Introduction Microbial polysaccharides ~-6 can be found intracellularly as reserve material (granules of starch or of glycogen), associated with the cell layers, or more or less free outside the cell as slime or free extracellular polysaccharides. Polysaccharides associated with the cell layers include: (a) lipopolysaccharides present in the cell wall of Gram-negative bacteria; (b) peptidoglycans of most bacterial cell walls in which polysaccharide chains are cross-linked via short peptide chains; (c) teichoic acids that are present in cell-walls and membranes of gram-positive bacteria; and (d) capsular polysaccharides (bound to the cell wall). * To whom correspondence should be addressed. Present address: Agriculture Canada, Centre de recherches alimentaires de SaintHyacinthe, 3600 boul. Casavant Ouest, Saint-Hyacinthe,Qurbec, J2S 8E3, Canada 198

Enzyme Microb. Technol., 1988, vol. 10, April

The 6-deoxysugars include aldoses such as 6deoxy-altrose, 6-deoxy-glucose (quinovose), 6-deoxymannose (rhamnose), 6-deoxy-gulose, 6-deoxy-idose, 6-deoxy-galactose (fucose), 6-deoxy-talose, 6-deoxyallose. The ketoses deoxysugars include 6-deoxypsicose, 6-deoxy-fructose, 6-deoxy-sorbose and 6deoxy-tagatose. Only three of these are commercially available (Figure 1). These are the 6-deoxy-glucose extracted from the cinchona bark, 7 the 6-deoxymannose, isolated mainly from arabic gum and also from bacterial cell wall, 8 the skin of rabbits, 9 or from the leaves of Solanum chacoensis, l° and finally there is the 6-deoxy-galactose, which occurs in seaweed 11 and in gum tragacanth. Another natural source of L-rhamnose includes spring sap of the maple sugar. 12 The industrial price of quinovose, fucose and rhamnose is estimated to be 200 000 $/kg, 5000 $/kg, and 130 $/kg respectively. The prices of these sugars as they are produced make them rather unattractive and do not stimulate their widespread use. Apart from the microbial synthesis of polysaccharides, other methods can be used to prepare deoxysugars. They include the photochemical reaction of O-acetylsugars in hexamethylphosphoric triamide (HMPT)/water 13 and the reduction of diol thiocarbonates with tributylin hydride) 4 Chemical synthesis of a rhamnose-containing polysaccharide has also been reported./5 It is beyond the scope of this paper to review these syntheses. This review is limited to microorganisms reported to produce polysaccharides or biosurfactants (surface active compounds) containing 6-deoxysugars. Culture conditions enhancing the microbial production of 6deoxysugar-containing polysaccharides have been examined, as well as the methods used to isolate and to hydrolyse the polysaccharides and to measure and to purify the 6-deoxysugars. The commercial applications of the 6-deoxysugars and 6-deoxysugar-cont~uning polysaccharides are discussed. © 1988 Butterworth Publishers

M i c r o b i a l p o l y s a c c h a r i d e s w i t h 6 - d e o x y s u g a r s : M. G r a b e r et aL Table 1

Microorganisms producing 6-deoxysugars (6-d-s) containing polysaccharides 6-d-s" free (F), cell-associated (C), cell wall (CW)

Species

References

Bacteria Acinetobacter calcoaceticus Acetobacter xylinum Actinomyces israelii Actinomyces viscosus Actinoplanes philippinensis Aerobacter cloacae Alcaligenes sp. Alcaligenes faecalis Amorphosporangium auranticolor Ampullariella regularis Arizona Arthrobacter carbozolum Arthrobacter simplex Azotobacter chroococcum Azotobacter indicum Azotobacter vinelandii Bacillus megaterium Bacillus polymyxa Bacillus oligonitrophilus Bacillus subtilis Bacterium cadaveris Beijerinckia mobilis Blastobacter viscosus Chromatobacterium prodigiosum Chromobacterium violaceum Chlorobium thiosulphatophilum Citrobacter freundii Corynebacterium floccumfaciens Corynebacterium insidiosum Corynebacterium midriganese Corynebacterium tritici Enterobacter sakazaki Escherichia alkalescens Escherichia coil Escherichia dispar Flavobacterium ugilinosum Klebsiella sp. Klebsiella aerogenes Klebsiella pneumoniae Lactobacillus bifidus Lactobacillus casei rhamnosus Malleomyces pseudomallei Marine bacterium Methylomonas methanica Methylococcus thermophilus Methylocystis parvus Mycobacterium album Mycobacterium tuberculosis Myxobacterium sp. Pseudomonas sp. Pseudomonas aeruginosa Pseudomonas atlantica Pseudomonas cepacia Pseudomonas elodea Pseudomonas hydrogenovora Pseudomonas maltophilia Pseudomonas solanacearum Rhizobium sp. Rhizobium cowpea Rhizobium japonicum Rhodospirillum rubrum Salmonella sp. Salmonella anatum Salmonella barilly Salmonella choleraesuis Salmonella champaign Salmonella dakar

(c, F) (CW) (F) F F,R F,R

(F) (F) (CW)

F R R R R R R F R

(F) (F) (F) (F) (F) (CW) (F) (F)

F

(F)

R R R R R,F R R R,F

(CW) (F) (F) (CW) (CW) (CW) (CW) (F) (F) (F) (F) (F) (CW) (C, CW) (CW) (F) (F, C) (CW) (C) (F) (CW)

F R

F F,R

F F,R R F

L R R F R R,F R

(CW) D-R F,R R R D-R R R R F,R R R R F,R R R

(F, CW) (CW) (F) (CW) (F) (F) (CW) (F) (C, F) (F) (F) (CW) (CW)

F F

(F) (F)

F R

25 4, 72 115 4, 78 56 8, 4O, 79 4, 8O 8, 81 56 56 82 47 4, 83 4 4, 7O 39, 78 78, 84 4, 35 115 4, 85 8 71 86 8 115 8 51 4 4, 78 4 4 8O 8 40, 43, 46, 47, 78, 79 8 4 33, 35, 45, 63, 65, 78, 87, 88 45, 78 4, 8O 4, 35 26 115 4 4 89 4, 109 78 115 4 8, 34, 42, 80, 90 18, 78, 91 4 92 4 4 4, 78 78, 93, 94 4, 67, 68, 69, 95 4 4, 78, 96 8 78 97 4O 4O 82 82

E n z y m e M i c r o b . T e c h n o l . , 1988, v o l . 10, A p r i l

199

Review Table

1

Continued 6-d-s a free (F), cell-associated (C), cell wall (CW)

Species

Salmonella enteritidis Salmonella gallinarum Salmonella grimpensis Salmonella typhimurium Salmonella paratyphi B Salmonella poona Salmonella wandsworth Serratia marcescens Serratia piscatorum Shigella boydii Shigella dysenteriae Shigella flexneri Streptococcus sp. Streptococcus bovis Strepto#occus mitior Streptococcus pneumoniae Streptococcus sanguis Streptococcus sobrinus Xanthomonas sp. Xanthomonas campestris Xanthomonas juglandis Yersinia pseudotuberculosis Yersinia enterolitica

F R R R R, F R R R, T R R, F R R R R R F A

Absidia cylindrospora Alternaria solani Armillaria mellea Botrytis cinerea Candida bogoriensis Ceratocystis ulmi Ceratocystis stenoceras Cladosporium tricoides Coriolus hirsutus Coriolus versivolor Cryptococcus elinorii Flamnulina velutipes Fusarium solani Mucor mucedo Mucor racemosus Polysporus fomentarius Polyporus ignarius Polyporus tuberaster Rhizopus nigricans Rhodotorula glutinis Sporothrix schenkii Tremella fusiformis

F R F R R, F R R R F F F F R F D- F F F F F R, F R F

Chlorella pyrenoidosa Rhodella maculata

R R

F, R R

(F) (CW)

F F

(F) (F)

(F) (CW) (CW) (CW) (C) (CW) (C) (CW) (F) (CW) (F)

References 40, 98 8 35 40 40 35 82 78 4, 8 99 78 78 54, 100, 101, 108 52, 53 102 60, 67, 78, 103, 104 102 103 105 50 4 78 78

Fungi (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F) (F)

4 4 4, 78 4 4 106 4, 78 4 4 4 4 115 4 4 4 4, 78 4 4 4 4, 62, 107 4, 78 4

(CW) (F)

57 8, 66

Algae

6-d-s: 6-deoxysugars, A: 6-d-L-altrose; D-R:D-rhamnose; D-F: t~-fucose; F: L-fucose; R: L-rhamnose; T: 6-d-L-talose. Blank spaces indicate that the 6-d-s has either not been identified and/or that its position has not been described

"

Distribution of microorganisms producing 6-deoxysugar-containing polysaccharides Microorganisms producing polysaccharides containing 6-deoxysugars are either Gram-positive or Gram-negative, aerobes or anaerobes, bacteria, algae or fungi of 200

Enzyme Microb. Technol., 1988, vol. 10, April

different metabolic capabilities so that the specific functions o f this type of polysaccharide are difficult to define. Within a single species or genus, different 6-deoxysugars as well as different cellular location have been observed. These kinds of variations are well exemplified by Acinetobacter calcoaceticus, Escherichia coli, Klebsiella and Pseudomonas (Table 1).

Microbial polysaccharides with 6-deoxysugars: M. Graber et aL CH3

HO]

0

OH

(0

H 9,/II--'--q OH

OH

detected macroscopically since some of them have a mucoid or watery aspect. ]9 A screening procedure based on the ability of some polysaccharides to form stable complexes with water-soluble dyes such as aniline blue 2° remains to be investigated. In some bacteria, exopolysaccharide synthesis appears to share common precursors and cofactors with cell wall and protein syntheses, hence competition for these intermediate would take place and the rate of polysaccharide synthesis would be influenced by the growth rate. E] The use of antibiotics such as tetracycline, streptomycin and chloramphenicol to uncouple the production of a polysaccharide from other syntheses 22 could permit the selection of high producing strains. Bacteriophage resistance has also been used as selective agents of mucoid strains. 23

OH

O0 J~OOH

"'HJ

OH

010 Figure 1 (I): 6-deoxy-D-glucose or quinovose; (11): 6-deoxy-Lmannose or rhamnose; (111):6-deoxy-L-galactose or fucose

Culture conditions for screening microorganisms and factors promoting their production Screening of microorganisms To date, no screening strategy has been elaborated to select microorganisms producing 6-deoxysugarcontaining polysaccharides. The primary aim of this section is to give some available information, which although applicable to polysaccharides in general, could be used to design such a strategy. Carbohydrate-rich environments such as effluents from the sugar, paper, food industries, breweries and wastewater plants are sources susceptible to contain microorganisms producing polysaccharides. ]6 Microorganisms present in sewage activated sludge were found to produce rhamnose containing polysaccharides. ]7 Isolation of microorganisms capable of utilizing petroleum derivatives could be another alternative to search for polysaccharide-producing organisms, since those microorganisms produce bioemulsitiers when incubated in the presence of such substrates and some bioemulsifiers contain rhamnose.18 The isolation of the microorganisms can be performed on rich complex media or on selective media using culture conditions known to promote the production of polysaccharides (see below). The colonies of microorganisms producing polysaccharides can be

Factors promoting production Specific culture conditions affecting the composition of sugars in a given 6-deoxy-sugar-containing polysaccharide produced by a specific strain have rarely been described. 24-z8 A number of physiological conditions are known to influence the production of polysaccharides.16'29'3°'31 They include the source and concentration of carbon 24'25'26'32'33'34 nitrogen, 32'34 and phosphorous 34 carbon-nitrogen ratio 34'35 type and concentration of ions,34 dilution rate 26'36 aeration rate, incubation temperature, p H , 34'36 age of the culture, 37 and nutrient limitation. 34 A survey of the above described studies revealed that the culture conditions promoting the production of microbial polysaccharides vary from one microorganism to another. The only enhancing parameter that appears to be constant is the cultivation of the polysaccharide-producing microorganisms in a medium having a high carbon nitrogen ratio.

Recovery of polysaccharides Microbial polysaccharides containing 6-deoxysugars make up a group in which structural and chemical composition variations are boundless. For this reason, the conditions used for the extraction differ more or less for each polysaccharide. Nevertheless the extraction methods are alike with regard to the cellular location of the polysaccharide. The extracellular, capsular, and cell wall polysaccharides are considered one after another. The selection of the extraction methods described in this section does not imply that they are selective for such polysaccharides. For an excellent general review on the recovery of microbial polysaccharides, the reader is referred to Smith and Pace. 3s

Extraction of exopolysaccharides Exopolysaccharides are readily solubilized and can be simply isolated in rather homogenous form by centrifugating the cells which are then discarded. The speed

Enzyme Microb. Technol., 1988, vol. 10, April

201

Rev~w and duration of centrifugation depend on the viscosity of the polysaccharides which is very changeable) 9 The polysaccharides can be then precipitated from the supernatant by addition of a lower alcohol e.g., 3 to 5 volumes of cold, often acidified, ethanol 25'35'4°-44 or propano142 or 1.5 to 4 volumes of acetone. 25'35'4°'42'45 The polysaccharides can be finally recovered by centrifugation or filtration as a white powder.

Extraction of capsular polysaccharides Polysaccharide must first be dissociated from the cells. The method used depends on the nature of the association between the cells and the polysaccharide. When the association is weak, centrifugation is sufficient to achieve the separation and the extraction can be carried out as described for the exopolysacchari d e s . 46-48 Drastic conditions of extraction are needed when the associations are stronger. Alkaline treatment with sodium hydroxide followed by an incubation of one hour and neutralisation can permit the recovery of the capsular polysaccharides after centrifugation and alcohol precipitation. 35,41 Heat treatment such as boiling the cells suspension for 15 rain in water, 35 or heating in saline solutions at 6 0 ° C 46'47 o r in a mixture of phenol-water at 6 5 ° C 47 o r sonification of the cell suspensions 48 can be used.

Extraction of cell wall polysaccharides The lipopolysaccharides of Gram-negative bacteria can be extracted using a mixture of phenol-water at 7 0 ° C . 49 Depending on their degree of solubility, lipopolysaccharides can be recovered either in the a q u e o u s phase 46 or in the phenol phase. 5°'51 Cell wall polysaccharides can also be extracted by hydrolysis with boiling HCI followed by centrifugation52'53 or by extraction with KOH. Other methods involve cell disruption with a Braun homogenizel~4-56 or a Mickel disintegrators7 and separation of the cell wall by ultracentrifugation. The cell walls are then treated with proteinases and ribonucleases before their extraction with formamide at warm temperature.

Purification of polysaccharides Most of the procedures for polysaccharides purification begin with the following stages. Concentration of the polysaccharide is carried out through a series of selective precipitations with lower alcohols or acetone. Other processes that permit elimination of a part of the proteins are often inserted between two precipitations; they include the use of either chloroform, trichlorotrifluoroethane, or proteinases. Removal of contaminating nucleic acids is performed with the help of ribonucleases or by precipitation with trichloracetic acid. Selective precipitation of polysaccharide with alkaline copper reagents 49 as well as separation of neutral polysaccharides by gel filtration, or of acidic polysaccharides by ion-exchange chroma-

:)02 Enzyme Microb. Technol., 1988, vol. 10, April

tography, electrophoresis or the precipitation by cetyltrimethyl-ammonium bromide, can also be used.

Qualitative and quantitative determination of the 6-deoxysugars in polysaccharides Colorimetric method The method of Dische and Shettles 58 using sulphuric acid and cysteine hydrochloride is widely used and considered satisfactory for non-purified extracellular polysaccharides 25'42'59 or purified cellular polysaccharides. 54'6° Reported interfering substances include lipopolysaccharides,51 hexoses and proteins. 33 One advantage of this method is that the polysaccharides do not need to be hydrolysed before the assay as they are hydrolysed during the assay. The concentration of 6-deoxysugars can also be determined according to the method of Dubois et al. 61 which is based on the fact that sugars give a yellow-orange color when treated with phenol and concentrated sulfuric acid after heating. The assay is carried out by measuring the absorbance at 480 nm and comparing the results with standard curves obtained under the same conditions.

Chromatographic methods These methods necessitate hydrolysis and neutralization of the polysaccharides. The conditions of hydrolysis are empirically chosen to obtain the highest reducing value. Most hydrolysis are performed at 100°C with sulfuric acid at concentrations ranging from 0.1 to 2.0 N during 2 to 16 h. Neutralization can be achieved with baryum carbonate or baryum hydroxide followed by the elimination of the precipitates by filtration or by using ion-exchangers such as Amberlite I R A 410 40 or Dowex 1.51 Demineralisation such as removal of baryum ions is achieved by passing the sample through a column filled with Dowex 50 41,62 or Amberlite IR I20. 63'64 Hydrolysis with hydrochloric acid can be used at concentrations ranging from 0.1 to 2.0 N during 2 to 5 h. The hydrolysates can then be neutralized with baryum carbonate, followed by filtration6° or by passing through a column Dowex 1. 59 Evaporation to dryness of the hydrolysed sample and redissolution in distilled water, have also been used. 4J Other acids such as trifluoroacetic acid (1.0 to 2.0 N, for 1 to 10 h at 100°C), 65'64 formic acid 66 and successive hydrolysis with two acids have also been reported. In this case, a first acid such as formic acid 33'35 or trifluoroacetic a c i d , 64'67 is used for 20 rain at 100°C and eliminated by evaporation. Sulfuric acid is then used at 100°C. Methanolysis can be used before gas liquid chromatography54'62'64 on cell walls fractions54 or on isolated polysaccharides. 62'64 It is carried out with 3% methanolic hydrogen chloride at 100°C for 3 h. 54 or 16 h. 62 The methanolic layer containing methylglycosides is neutralized on a column of Amberlite IRA4B (OH) and concentrated to dryness before gas liquid chromatography .54

Microbial polysaccharides with 6-deoxysugars: M. Graber et aL Separation between the neutral and the acidic sugars can be achieved by passing them through a column containing Dowex 1-2x41 or Amerlite IR 45, which fix the acidic sugars.

Paper chromatography The chromatographic papers which can be used are the Whatman No. 1 or 3.63'65'68 Elution is realized with solvents system composed of water, an organic watermiscible polar solvent (ethanol, pyridine, acetic or formic acid) and an organic water miscible non polar solvent (n-butanol, ethyl acetate). The sugars are detected with one of the following reagents usually after heating 5-10 min at 100-110°C: p-anisidine hydrochloride, 42'6° aniline hydrogen phosphate, 69 aniline hydrogen phtalate, 7 saturated aqueous aniline oxalate 33'35 alcaline silver nitrate.

Thin-layer chromatography Thin-layer chromatography can be used for the qualitative determination of sugars. 4°'62'46,51 It is performed on Kiesel-gel G plates 62 or on MN 300 cellulose plates. 4°'51 The solvents used include the following mixtures: butanol-pyridine-water (4 : 6 : 3) 13'18 or (5 : 1 : 2)51 and butanol-isopropanol-water (5 : 3 : 1). 62 The development takes about 2 h. 62 Sugars are detected by spraying with alcaline silver nitrate 5~ or 5% methanolic sulfuric acid. 62

Gas-liquid chromatography Gas chromatography identification and quantitation of 6-deoxysugars can be done after conversion 7° of the sugars into their alditol acetates 51'53,55,63,64,7~ or conversion 72 into the corresponding O-trimethylsilylether. 54'62'67 The glass and stainless steel columns used to separate the converted sugars into their alditol acetates (2 to 3 meters long with an outer diameter ranging from 3 to 6 mm) contain 3% 5L53'55 to 5% 24 of ENMS-M or of OV 22555'71 on Gas Chrom Q (100-200 mesh) 51'53'63'64'71 or on Supelcoport (80-100 mesh). 55 The glass and stainless steel columns (2.0 m long, 3 mm outer diameter) used to separate the converted sugars into their corresponding O-trimethylsilylether contain 5%, 54 1 0 % 67 o r 1 5 % 62 of UCON LB 550 X on Gas Chrom CLM 54 or Chromosorb W. 62'67

Identification of 6-deoxysugars by crystallization The crystallization followed by the measure of the melting point can be used to confirm the identification of the 6-deoxysugars. Among the different forms of crystalline 6-deoxysugars that can be obtained are the aldose phenylosazone derivatives, 5° the phenylhydrazone derivatives 33 and the p-nitroaniline aldose derivatives.

Uses of 6-deoxysugar-containing polysaccharides The commercial usefulness of most industrial polysaccharides is based on their ability to alter the basic properties of water. In this respect, they can be used as stabilizers, suspending agents, dispersants, thickeners, film-forming agents, water-retention agents, coagulants, colloids, lubricant or friction reducers. Polysaccharides are thus used extensively in laundry products, textiles, adhesives, paper, paint and foods. 6-Deoxysugar-containing polysaccharides that have potential industrial applications include gellan, S-130, S-194, 5 S198, PS7, PS10, PS21 and P 8 5 3 . 6'74 The uses of these 6-deoxysugar-containing polymers as a whole include gelling agent for microbiological media, 75 structuring, texturizing, stabilizing agents in food systems, drilling and suspending fluids, and thickeners in the paint industry.5.74Some 6-deoxysugar-containing polysaccharides have bioemulsifying activity.18'25 However, the role of the 6-deoxysugars is not well

HO

O

H3C

CH3

HO

0

H 3C" ~

H

00

Liquid chromatography The 6-deoxysugars can be determined using h.p.l.c. The sugars can be detected using ion chromatography column and pulsed amperometric detector 24 or using refractometry and h.p.l.c, column such as Aminex or Bio-Sil (Bio-Rad, California, USA). The 6-deoxysugars can also be separated using low pressure liquid chromatography. They can be eluted in columns filled with ion-exchange resins such as Dowex as borate c o m p l e x e s .73

HsC2/~C2Hs

(l i a) Figure 2 (I): 4-hydroxy-2,5-dimethyl-2,3-dihydrofuran-3-one (Furaneol), fruity flavor e.g., strawberry. (11): 4-hydroxy-5methyl-2H,2,3-dihydrofuran-3-one, boiled beef flavor; fill): 4hydroxy-2,5-diethyl-2,3-dihydrofuran-3-one, meat flavor enhancer Enzyme Microb. Technol., 1988, vol. 10, April

203

Review Table 2

Substrates and conditions used for the production of Furaneol and its derivatives

Starting substrate

Reactants and conditions

Melting point of the product (°C)

Yield

References

6-deoxy-mannose

piperidine acetic acid ethanol aerobiosis

82-83

43

110

6-deoxy-fructose 6-deoxy-sorbose 6-deoxy-glucose 6-deoxy-mannose 6-deoxy-galactose

piperidine acetic acid ethanol anaerobiosis

79-80

65

76,77

fructose-1,6-phosphate

water water : methanol

77

14-41

111

fructose-l-phosphate

NaOH or KOH or piperidine acetate, palladium or coal, 15-60 psi

96

fructose-6-phosphate ribose-5-phosphate

sodium acetate acetic acid water

erythro-dihyd roxydiketone

tartaric aid

126-128

34

112

NaHCO3 NaHPO4 pentane

78-80

51

113

methylmagnesium chloride tetrahydrofuran ne trifluoroacetic acid : water (9 : 1) piperidine acetic acid, water

73-78

48

114

understood with regard to the properties they confer to these polysaccharides. The 6-deoxysugar-containing polysaccharides can also be regarded as a source of 6-deoxysugars. For instance, some 6-deoxyhexoses such as rhamnose and fucose, can be used as substrates in the chemical synthesis of Furaneol, 76'77trade name for 2,5 dimethyl-4-hydroxy-2,3-dihydrofuran-3one, of proprietary Firmenich, and can be used as flavoring agent. Although intensive research on biosynthesis of natural Furaneol is being conducted, at the present time this compound is mostly chemically synthesized from 6-deoxysugars (Table 2). The industrial price of Furaneol is estimated to be 100-200 US$/kg. Biochemical synthesis of Furaneol using enzymes and microorganisms could level up its cost, but it should increase and diversify its uses. Furaneol, a flavor principle with a powerful caramel-like flavor has found many applications in the food and beverage industry. By changing the radicals of the basic Furaneol molecule, the concentration of Furaneol and of others components such as short chain fatty acid esters, either the strawberry, pineapple, grapefruit, boiled beef flavors or others can be obtained (Figure 2).

204

77

Enzyme Microb. Technol., 1988, vol. 10, April

References 1 2 3 4

5 6 7 8 9 10 11 12 13 14 15

Kenne, L. and Lindberg, B. in The Polysaccharides, (Aspinall, G. O., ed.) Academic Press, New York, London. 1983, 2, 287-365 Painter, T.J. in The Polysaccharides (Aspinall, G. O., ed.) Academic Press, New York, London. 1983, 2, 196-286 Gorin, P. A. J. and Barreto-Bergter, E., in The Polysaccharides (Aspinall, G. O., ed,) Academic Press, New York, London 1983, 2, 366-411 Berthelet, D. et al. Bibliographique. Vol. I. Rapport du CERMAV pour le minist~re de l'industrie et de la recherche dans le cadre du programme mobilisateur "Essor des biotechnologies." April 1984 Sandford, P. A., Cottrell, I. W. and Pettitt, D. A., Pure and Appl. Chem. 1984, 56, 879-892 Paul, F., Morin, A., and Monsan, P. F. Bioteehnol. Adv. 1986, 4, 245-259 Freudenberg, K., Raschig, K. Bericht. 1929, 62, 373-383 Salton, M. R. J. Biochim. Biophys. Acta. 1960, 53, 449-452 Maluwista, I. and Davidson, E. A. Nature 1961, 192, 871-872 Kuhn, R. and LOw, I. Chem. Ber. 1961, 94, 1088-1095 Hockett et al. J. Arner. Chem Soc. 1939, 61, 1658 Adams, G. A., Bishop, C. T. and McDonald, I. J. Can. J. Biochem. Physiol. 1959, 37, 507-510 Pete, J-P. and Portella, C. Synthesis 1977, 11, 774-776 Barton D.H.R. and Subramanian, R.J.C.S. Chem. Comm. 1976, 21, 867-868 Wessel, H-P., and Bundle, D. R., J. Chem. Soc. Perkin Trans. 1985, I, 2251-2260

Microbial polysaccharides with 6-deoxysugars: M. Graber et aL 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57

Lawson, C. J. and Sutherland, I. W. in Economic Microbiology, (Rose, A. H., ed.), 1978, 2, 327-392 Rideau, J. P. and Morfaux, J. N. Water Res. 1976, 10, 999-1003 Guerra Santos, L. H., Dissertation ETH No. 7722, Swiss Federal Institute of Technology, Z0rich, 1985 Govan, J. R. W. J. Antimicrob. Chemother. 1976, 2, 215-217 Nakanishi, I. et al. Carbohydr. Res. 1974, 32, 47-52 Sutherland, I. W. Surface Carbohydrates o f the Prokariotic Cell, Academic Press, London, 1977, 472 pp. Rubinovitz, C., Gutnick, D. L. and Rosenberg, E. J. BacterioL 1982, 152, 126-132 Martin, D. R. J. Med. Microbiol. 1973, 6, 111-118 Bryan, B. A., Linhardt, R. J. and Daniels, L. Appl. Environ. Microbiol. 1986, 51, 1304-1308 Kaplan, N. and Rosenberg, E. Appl. Environ. Microbiol. 1982, 44, 1335-1341 Wicken, A. J. et al. J. Bacteriol. 1983, 153, 84-92 Morin, A., Duchiron, F., and Monsan, P. F. J. Biotechnol. 1987, 6, 283-306 Graber-Gubert, M., Morin, A., and Monsan, P. Syst. Appl. Microbiol. 1988, 10 Bull, A. T. J. Appl, Chem. Biotechnol. 1972, 22, 261-292 Ellwood, D. C. and Tempest, D. W. Adv. Microb. Physiol. 1972, 7, 83-117 Troy II, F. A. Ann. Rev. Microbiol. 1979, 33, 519-560 Cohen, G. H. and Johnstone, D. B. J. Bacteriol. 1964, 44, 1335-1341 Wilkinson, J. F., Dudman, W. F., and Aspinall, G. O. Biochem. J. 1955, 59, 446-451 Williams, A. G. and Wimpenny, J. W. T. J. Gen. Microbiol. 1977, 102, 13-21 Dudman, W. F. and Wilkinson, J. F. Biochem. J. 1955, 62, 289-295 Linzer, R., Campbell, L. K. and Know, K. W. Infect. Immun. 1984, 44, 76-81 Bailey, R. W., Greenwood, R. M. and Craig, A. J. Gen. Microbiol, 1971, 65, 315-324 Smith, I. H., and Pace, G. W. J. Chem. Tech. Biotechnol. 1982, 32, 119-129 Sutherland, I. W. Adv. Microbiol. Physiol. 1972, 8, 143-213 Grant, W. D., Sutherland, I. W., and Wilkinson, J. F. J. Bacteriol. 1969, 100, 1187-1193 Cohen, G, H. and Johnstone, D. B. J. Bacteriol. 1964, 88, 329-338 Williams, A. G,, and Wimpenny, J. W. T. J. Gen. Microbiol. 1977 102, 13-21 Sapelli, R. V., and Goebel, W. F. Proc. Natl. Acad. Sci. 1964, 52, 265-271 Goebel, W. F. Proc. Natl. Acad. Sci. 1963, 49, 464-471 Nimmich, W. Z. AIlg. Mikrobiol. 1979, 19, 343-347 Jann, B., Jann, K., and Schmidt, G. Eur. J. Biochem. 1970, 15, 29-39 Jann, K., Jann, B., and Schneider, K. F. Eur. J. Biochem. 1968, 5, 456-465 Glaudemans, C. P. J., and Treffers, H. P. Carbohydr. Res. 1967, 4, 177-181. Whistler, R. L. Methods in Carbohydrate Chemistry, vol. 1, Academic Press, New York, London, 1965 Hickman, J., and Ashwell, G. J. Biol. Chem. 1966, 241, 1424-1428 Rudolf, A. R. and Wheat, R. W. J. Bacteriol. 1968, 95, 2035-2043 Pazur, J. H., Dropkin, D. J., and Forsberg, L. S. Carbohydr. Res. 1978, 66, 155-166 Pazur, J. H. et al. Arch. Biochem. Biophys. 1976, 176, 257-266 Shioiri-Nakano, K., Tadokoro, I., and Kudo, M. Jap. J. Exp. Med. 1966, 36, 563-576 Bleiweis, A. S., Karakawa, W. W. and Krause, R. M. J. Bacteriol. 1964, 88, 1198-1200 Szaniszlo, P. J., and Gooder, H. J. Bacteriol. 1967, 96, 2037-2047 Northcote, D. H. and Goulding, K. J. Biochem. J. 1958, 70, 391-397

58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97 98 99 100 101 102

Dische, Z. and Shettles, L. B. J. Biol. Chem. 1948, 175, 595-603 Kang, S., and Markowitz, A. J. Bacteriol. 1967, 93, 584-591 Miyazaki, T., and Yadomae, T. Carbohydr. Res. 1971, 16, 153-159 Dubois, M. et al. Anal. Chem., 1956, 28, 350-356 Fukagawa, K. et al. Agric. Biol. Chem. 1974, 38, 29-35 Joseleau, J. P., Lapeyre, M., and Vignon, M. Carbohydr. Res. 1978, 67, 197-212 Dutton, G. G. S. and Paulin, M. Carbohydr. Res. 1980, 87, 107-117 Joseleau, J. P., and Marais, M. F. Carbohydr. Res. 1979, 77, 183-190 Fareed, V. S., and Percival, E. Carbohydr. Res. 1977, 53, 276-277 Roy, N., Carroll W. R., and Glaudemans, C. P. J, Carbohydr. Res. 1970, 12, 89-96 Kennedy, L. D. Carbohydr. Res. 1980, 87, 156-160 Bailey, R. W., Greenwood, R. M., and Craig, A. J. Gen Microbiol. 1971, 65, 315-324 Sawardeker, J. S., Slokener, J. H. and Jeanes, A. R. Anal. Chem. 1965, 37, 1602-1604 Cooke, A. A., and Percival, E. Carbohydr. Res. 1975, 43, 117-132 Savidge, R. A. and Colvin, J. R. Can. J. Microbiol. 1985, 31, 1019-1025 Floridi, A. J. Chromatog. 1971, 59, 61-70 Cottrell, I. W. in Fungal Polysaccharides (Sandford, P. A., and Matsuda, K., eds. Amer. Chem. Soc. Symp. Ser. 1980, 126, pp. 251-270 Shungu, D. et al. Appl. Environ. Microbiol. 1983, 46, 840845 Matsui, M. Ogawa, T. and Tagaki, K. Jap. Pat. Jpn. Kokai Tokkyo Koho 79 19962 (1979) Wong, C.-H., Mazenod, F. P. and Whitesides, G. M. J. Org. Chem. 1983, 48, 3493-3497 Aspinall, G. O. The Polysaccharides, vol. I, I1,111, Academic Press, New York, 1982 Heildelberger, M. et al. J. Bacteriol. 1968, 95, 2415-2417 Voelskow, H. and Schlingmann, M. Ger. Pat. 3300633 (1984) Lipt~ik, A. Carbohydr. Res. 1982, 107, 300-302 Luderitz, O. et al. J. Bacteriol. 1967, 93, 1681-1687 Hagiwara, S. and Yamada, K. Agric. Biol. Chem. 1970, 34, 1283-1295 White, P. J. and Gilvarg, C. Biochemistry, 1977, 16, 24282435 Morita, N., Takagi, M., and Murao, S. Bull. Univ. Osaka Pref. Ser. B, 31, 27-41 Loginova, N. V. and Trotsenko, Y. A. Prikl. Biokhim. Mikrobiol. 1980, 16, 331-334 Choy, Y. M., and Dutton, G. S. Can. J. Chem. 1973, 52, 684-687 Dutton, G. G. S. and Lim, A. S. Carbohydr. Res. 1985, 144, 263-276 Grinberg, T. A., Kosenko, L. V. and Malashenko, Y. R. Mikrobiol. Zh. (Kiev), 1984, 46, 22-26 Syldatk, C. et al. Naturforsch 1985, 40C, 61-67 Asonganyi, T. M., and Meadow, P. M. J. Gen. Microbiol. 1980, 117, 1-7 Knirel, Y. et al. Bioorg. Khim. 1980, 6, 1851-1859 Liao, Y. M., and Hsu, S. T. Chung-hua Nung Yeh Yen Chi, 1980, 29, 265-271 Akiyama, Y. et al. Agr. Biol. Chem. 1985, 49, 1193-1194 Jackson, L. K. et al. Carbohyd. Res. 1980, 82, 154-157 Singh, A., Vadhera, S., and Singh, R. 1977, 15, 404-406. Milev, M., and Slavkov, I. 2nd Salmonelite Salmonelozite Bulg. Nat. Konf. 1971, 141-149 Minev, M. 2nd Salmonelite Salmonelozite Bulg. Nat. KonJ~ 1971, 225-230 L'lov, V. L. et al. Biorg. Khim. 1980, 6, 1842-1850 Krause, R. M. Bacteriol. Rev. 1963, 27, 369-380 Heymann, H., Manniello, J. M., and Barkulis, S. S. J. Biol. Chem. 1963, 238, 502-509 Price, T. et al. J. Gen. Microbiol. 1986, 21, 189-197

Enzyme Microb. Technol., 1988, vol. 10, April

205

Review 103 104 105 106 107 108 109

206

Linzer, R., Sreenivasulu, M., and Levine, M. J., Inf. lmmun. 1985, 50, 583-585 Barker, S. A. et al. Carbohydr. Res. 1986, 1, 393-399 Matyshevskaya, M. S. et al. 1985, 47, 30-33 Ballou, C. Adv. Microbiol. Physiol. 1976, 14, 93-158 Fukagawa, K. et al. Nippon Nogei Kagaku Kaishi, 1973, 47, 651-653 McCarty, M., and Lancefield, R. C. J. Exp. Med. 1955, 102, 11 Hou, C. T., Laskin, A. I., and Patel, R. N. Appl. Environ. Microbiol. 1978, 37, 800-804

Enzyme Microb. Technol., 1988, vol. 10, April

110 111 112 113 114 115

Hodge, J. E., Fischer, B. E., and Nelson, E. C. US Pat. 2 936 308 (1963) Whitesides, G., and Mazenod, F. P. Eur. Pat. 0090150 (1983) Peer, H.G.,andVanDenOuweland, G . A . M . Recueil, 1968, 87, 1017-1020 Bfichi, G., Demole, E., and Thomas, A. F. J. Org. Chem. 1973, 38, 123-125 Briggs, M. A., Haimes, A. H., and Jones, H. F., J. Chem. Soc., 1985, 4, 795-798. Loiseleur, J. Techniques de Laboratoire, Vol. I, Fascicule 2, Masson, Paris, 1963