Appl Microbiol Biotechnol (1992) 36:604-610

Mierobiology Biotechnology © Springer-Verlag 1992

Microbial modification of sugars as building blocks for chemicals E. Stoppok, K. Matalla, and K. Buchholz Institut for landwirtschaftliche Technologic und Zuckerindustrie an der TU Braunschweig, Langer Kamp 5, W-3300 Braunschweig, Federal Republic of Germany Received 16 July 1991/Accepted 21 October 1991

Summary. Investigations on the microbial modification o f sucrose to the corresponding 3-keto-derivative were carried out with resting cells of Agrobacterium tumefaciens N C P P B 396. This highly specific oxidation to yield the 3-keto-derivative has been analysed kinetically with varying substrate and cell mass concentrations. The formation o f the corresponding 3-keto-derivative d e p e n d e d strongly on the reaction time and the aeration rate, and was maximal at aeration rates up to 11.5 volume air/cultivation volume per minute with resting cells. The product formation increased with increasing substrate concentrations. However, the product yield was maximal at substrate concentrations below 20 g/1. Data pertaining to the production of active cell mass as well as for maximal 3-keto-derivative formation are presented in this paper. Also included are some applications for these derivatives.

Introduction Disaccharides, although available on a ton scale in very high purity and for a reasonable price, are not yet being used on a large scale as chemcials. The main reason is the high polyfunctionality o f the molecules, which hinders selective reaction routes such as those with sucrose. In contrast to sucrose, the 3-keto-derivative has at least one specific site for selective chemical synthesis and thus offers promising potential as a starting material for carbohydrate-based products. The microbial formation of 3-keto-glycosides was first described by Bernaerts and De Ley (1958) using a microorganism that was later identified as Agrobacterium tumefaciens (Bernaerts and De Ley 1963). Use of this microbial oxidation reaction was extended to various sugars by different groups (Bernaerts and De Ley 1960; Fukui and Hochster 1963; Hayano and Fukui 1968; Tyler and N a k a m u r a 1971; Kurowski and Darbyshire 1978), applying growing bacteria or resting cells

Offprint requests to: E. Stoppok

of A. tumefaciens or the purified enzyme (Grebner and Feingold 1965; Hayano and Fukui 1967; van Beeumen and De Ley 1975). The flavin adenine dinucleotide-dependent inducible enzyme, responsible for this oxidation reaction has been identified as hexopyranoside cytochrome c: oxidoreductase by van Beeumen and De Ley (1968). H a y a n o and Fukui (1967) proposed use of the trivial name glucoside 3-dehydrogenase, which was also utilized by some authors. Figure 1 shows the oxidation reaction with the above-mentioned enzyme exemplified with sucrose as the substrate. An advantage of using microbial cells for the production o f 3-keto-sugars is that it provides favourable conditions for the transfer of the reaction to a technical scale. For this reason all reactions occur in aqueous systems and no protecting groups are required, thereby facilitating isolation and purification of the product. Although keto-sugars are excreted to some extent into the culture broth in batch fermentations, the use o f resting cells with a high enzymatic activity was preferred and provided the advantage of achieving higher product yields via use of higher cell densities in the reaction mixture. Thus, the production o f active cell mass and the product formation phase (oxidation reaction) can be optimized separately. With respect to technical development as well as for preparation purposes it is essential to achieve several goals: high product yield, high product concentration and favourable kinetics. Reported herein are investigations concerning the above-mentioned aspects that have not been considered previously.

Materials and methods Microor#anism. Various strains ofA. tumefaciens are able to oxidize sucrose to 3-keto-sucrose. Among all the tested isolates strain NCPPB 396 (National Collection of Plant Pathogenic Bacteria, Harpenden, UK) was selected, as it had been shown to yield the best keto-sucrose formation. Cultivation. The culture was maintained on a medium consisting of 5 g/1 of peptone, 3 g/1 of meat extract and 15 g/1 of agar agar (deionized water).

605

HOH

HO

OH

OH.

OH HO o-D-glucopyranosyl-(l--=--2)~-D-fructofuranos~d

o-O-ribo-hexopyranosyl-3-ulose-(1--~-2)B-O-fructofuranosid

3-KETOSUCROSE

SUCROSE G-3-DH / FAD ATP--~

G-3-DH / FADHz

~ H20

-ADP 11202

Fig. 1. Oxidation of glucosides by Agrobacterium tumefaciens exemplified by sucrose: G-3-DH, glucoside 3-dehydrogenase; FAD, flavin adenine dinculeotide; ETP, electron transport pathway

The culture medium of Kurowski and Darbyshire (1978) was modified and consisted of 0.9 g/l urea, 0.5 g/1 yeast, 0.15 g/1 MgSO4.7H20, 0.025g/1 CaC12.2H20, 0.01g/1 FeSOa.7H20, 0.16 g/1 citric acid, 5.4 g/1 KH2PO4, 10.8 g/1 Na~HPOa.2H20 and 20 g/I sucrose. The pH of the medium was adjusted to 7.0 prior to sterilization. For inoculation of precultures, fresh cells from agar slants were transferred to 100 ml medium in 500-ml erlenmeyer flasks. These were incubated for 40-44 h at 27 ° C on a rotary shaker at 100 rpm. The main culture was harvested after a cultivation time of 20 h.

Fermentation. Fermentations were carried out in a 20-1 Biostat E (B. Braun, Melsungen, FRG) with a working volume of 141. Air was sparged into the culture broth with an initial aeration rate of 0.125 vvm (volume air/cultivation volume per minute) and was increased to 0.5 vvm 4 h later. A stirrer speed of 400 rpm was maintained. The temperature and pH were the same as for cultivations in Edenmeyer flasks.

oir nte

outlet~ J

vessel for wQter sc[tLit QtiOr~ Suspension of b(]ct eriol cells with sucrose

. I

Reaction mixture. Cells were harvested when the activity of the hexopyranoside c:oxidoreductase was maximal (20 h with edenmeyer flasks, 15-20 h in bioreactors) and washed twice with 0.1 M phosphate buffer, pH 7.0. For most experiments these resting cells were incubated at a temperature of 27° C with a cell mass concentration of 50 g bacterial wet weight (BWW)/1 and 50 g/1 sucrose at pH 7.0. Small amounts of cells derived from shaking flasks or fermentation samples were incubated in 100-ml edenmeyer flasks filled with 40 ml reaction mixture. Investigations on the optimization of product formation were performed in stirred reaction vessels with a working volume of 400 ml. These reactors were equipped with an aeration system and pH-control (Fig. 2). For storage of resting cells a concentrated cell suspension was stripped with nitrogen to prevent loss of activity by oxygen and then kept at 4°C until use (max. 5 days).

Assay. The activity of the hexopyranoside c:oxidoreductase was calculated from the formation of 3-keto-sucrose according to the method described by Fukui and Hayano (1969). A molar extinction coefficient of E=6.5 x 103 M-1 cm -1 was used. Sucrose and fructose were estimated by HPLC, glucose by Boehringer (Mannheim, FRG) test set.

of ~ir

~ir

,

F~g. ~. Reactor for the oxidation of saccharides: R, ~; M, ~ ; ~,~

Results

Effect of cultivation conditions of A. tumefaciens on keto-sugar formation Various c u l t u r e m e d i a were tested for their i n f l u e n c e o n the g r o w t h a n d the ability o f A. tumefaciens to p r o d u c e 3-keto-sucrose a n d h e n c e d e h y d r o g e n a s e activity. It s h o u l d be p o i n t e d out that all c u l t i v a t i o n effects o n the o x i d a t i o n o f sucrose were tested b y the use o f resting cells o f A. tumefaciens. A m o n g all tested m e d i u m c o m p o n e n t s m a i n l y M n 2+ a n d Fe 2+ ions h a d a clear effect o n the g r o w t h a n d activity o f h e x o p y r a n o s i d e c : o x i d o r e d u c t a s e o f A. turnefa-

606 Table 1. Effect of Mn 2+ and Fe2+ ions on the growth a and 3-keto-sucrose formationb of Agrobacterium tumefaciens 20-

Ion addition (~tmol/1)

Bacterial wet weight

Yield (%)

(g/l) 15 ~ F~ 2+

0.0 3.6 18.0 36.0 180.0 360.0

3.6 4.8 7.4 6.8 8.4 10.0

37.3 36.4 34.3 24.0 19.3 16.9

M n 2+ 0.000 0.947 9.470 94.700

7.0 11.3 12.7 12.3

37.6 16.3 15.2 15.2

L

g=~ 5~

0 ~

202

" Bacterial wet weight was analysed after 20 h of growth in eden-

meyer flasks. The Fe 2+ ion concentration was varied in the culture medium as described in Materials and Methods; Mn 2+ ions were added to the culture medium together with 10 mg/1 FeSO4 b Cells were harvested after 20 h and used as resting cells for the production of 3-keto-sucrose. The oxidation was performed as described in Materials and methods with eflenmeyer flasks on a rotary shaker. The yield is the amount of 3-keto-suerose derived from the oxidation of sucrose under this conditions

-50

,~o 15• 30

~ loo ~_

'20

I.~

ciens. This effect was more distinct with M n z+ than with Fe z+ ions. A drastic decrease in 3-keto-sucrose formation was observed even at very low M n 2+ concentrations, whereas growth was improved (Table 1). The yield o f 3-keto-sucrose was in the range 24-33% when sucrose or glucose were used as the carbon source in the culture medium. It decreased to 5% when fructose was the sole carbon source, an effect most likely due to the inducible nature of the enzyme as deduced b y van Beeumen and De Ley (1968). Sucrose could also be replaced by molasses as an inexpensive carbon source without significant loss of 3-keto-sucrose formation. The best results were obtained when sucrose was replaced by 3% molasses as a carbon source. Studies with various nitrogen sources revealed that urea was superior to other nitrogen sources. In order to investigate the oxidation reaction o f sucrose to 3-keto-sucrose with resting cells, greater amounts o f cell mass were p r o d u c e d by fermentations u n d e r the conditions described above. Figure 3 presents a typical fermentation course o f A . turnefaeiens with sucrose as the carbon source. After a short lag p h a s e 3-keto-sucrose formation increased to a m a x i m u m at 18-20 h. At this time the a m o u n t of 3-keto-sucrose excreted into the culture m e d i u m was 7.1 g/l, which is equivalent to a 35% yield. As growth was not complete at this time, we assume that the microorganisms consumed the keto-sucrose and possibly further products f r o m keto-sucrose. Cells harvested at different cultivation times and used as resting cells reached a m a x i m u m dehydrogenase activity at 17-18 h. U p o n incubation at a cell concentration of 50 g / l with 50 g / l o f sucrose, a higher

1

5-

0

'

2'0

'

Cultivation

io

time

-

'

6o

[hi

Fig. 3. Fermentation course of A. tumefaciens NCPPB 396

p r o d u c t concentration of 21.7 g/l, corresponding to a yield of 43%, was reached.

Keto-sucrose formation with restin9 cells The experiments with resting cells were p e r f o r m e d with the reaction mixture described above. Although the enzyme reaction is possible with different electron acceptors (van Beeumen and De Ley 1975), in view o f the acceptance and technical feasibility o f further products f r o m the 3-keto-derivatives, only oxygen was used. To provide high product formation during the whole oxidation reaction, the initial substrate concentration used was at least 73 mM, which corresponds to 25 g/l. This concentration was more than 50-fold of the Km of the resting cells, which was estimated to be 1.3 mM f r o m a H a n e s plot ( p H 7.0, T 27 ° C) in Fig. 4. The above Km is in good accordance to the K m o f 2.1 mM for resting cells that was found by Fukui and H o c h s t e r (1964). With 2,6-dichlorophenol i n d o p h e n o l as the electron acceptor, van Beeumen and De Ley (1975) found a Km o f 4.1 mM for the purified enzyme ( p H 6.04, T 30 ° C) whereas H a y a n o and Fukui (1967) reported a Km of 0.38 mM for sucrose as the substrate ( p H 7.0, T 20 ° C).

607

Table 2. Influence of pH on 3-keto-sucrose formation at different incubation timesa pH

Yield (%) 2.5 h

O

5.8 6.4 7.0 7.6 8.2 8.5

E

J 5

10

15

20

25

Sucrose [mMl Fig. 4. Hanes plot for the determination of Km with sucrose as the

substrate. The reaction mixture contained 2 g/l of resting cells (bacterial wet weight) and 1-25 mM substrate. The incubation temperature was 27°C at pH 7.0 in 0.1 M buffer solution: s/v, o

~"

25

.25

~ o

2o

211 ~

~.

g,

'15

§ e-

~0

;

°'i

p

,g ~_

0

5

~ I~

2

4

6

8

10

12

1~

~

13

Reaction time [h]

Fig. 5. Time course of 3-keto-sucrose formation with resting cells of A. tumefaciens in aerated reactors: ©, sucrose; O, 3-ketosucrose; A, sum of sugars. The biocatalyst concentration (wet weight) and sucrose concentration were 25 g/1 each. The aeration rate was 6.0 vvm at pH 7.0

In Fig. 5 the typical time course of sucrose oxidation is shown. Product formation reached a maximum and then gradually, decreased. The sum of sucrose and 3-keto-sucrose decreased successively during the oxidation reaction, indicating a further reaction of 3-keto-sucrose and sucrose. This reaction may partially be caused by polysaccharide formation, as previously postulated by Bernaerts and De Ley (1960). These authors also concluded that two mechanisms apparently compete for the substrates, namely, hydrolysis and oxidation. Assuming these reactions are verified, the maximum of product formation can be regarded as a consequence of simultaneous formation and degradation reactions. Conflicting results have been published on the appropriate p H during 3-keto-sucrose formation. Fukui and Hochster (1963) found an optimum of glycoside formation at a p H of 8.6 and later recommended a working p H of 7.0 (Fukui and Hochster 1965). Tyler and Nakamura (1971) showed that the p H for 3-ketomaltose formation should be in the range 6.5-7.0. Con-

4.0 14.0 23.5 32.4 27.4 25.9

5.0 h 5.9 26.3 37.9 49.3 29.9 23.8

7.5 h 8.1 35.0 49.4 45.3 36.8 10.3

11.5 h

19.5 h

8.9 b 40.0 45.5 37.1 26.7 9.3

5.8 26.6 32.7 22.1 9.6 1.1

a Incubations were carried out in aerated reactors (400 ml reaction volume) with a biocatalyst concentration (wet weight) and sucrose concentration of 25 g/1 each. The aeration rate was 8.0 vvm and the pH in each reactor was kept constant by automatic titration of HC1 or NaOH b Bold type shows the maximal yield at the corresponding pH sequently, experiments were performed to study the effect of p H on 3-keto-sucrose formation with resting cells of A. tumefaciens NCPPB 396. The time course of 3-keto-sucrose yield was estimated for each p H to take into account that the 3-keto-sucrose formation runs through a maximum (Fig. 5). The results in Table 2 show that this maximum occurred at different reaction times depending on the pH. The highest yields of 3keto-sucrose at p H values of 7.6-8.5 were observed at the relatively short incubation times of 2.5 and 5 h whereas maximum yields at p H 5.8 and 6.4 were found at incubation times of 11.5 h. The best yield was obtained at p H 7.0 and 7.6. However, at p H values higher than 6.4, fructose formation was observed with a tendency to increase over longer reaction times. To facilitate isolation of 3-keto-sucrose, it may be advantageous to work at a p H of 7.0 or even below. The yield of 3-keto-sucrose also depends on the amount of biocatalyst used in the reaction mixture. This effect was studied by varying the BWW in the reaction mixture (in shaking cultures) from 10 g/1 to 100 g/1. A linear correlation of product formation and biocatalyst concentration was expected but was found only at small biocatalyst concentrations. The results in Fig. 6 show that maximum product formation occurred at the highest biocatalyst concentration but the specific rate of product formation decreased clearly with increasing cell mass. It is supposed that oxygen limitation in the shake flasks is at least one reason for the non-proportional increase of product formation with increasing biocatalyst concentration. Although the specific rate of product formation was higher at low cell mass concentrations the experiments were carried out at biocatalyst concentrations of 25-50 g/l to reduce the reaction times. Thus, less inactivation of the enzyme system is expected during the reaction course. The effect of initial substrate concentrations (in shaking flasks) on the time course of 3-keto-sucrose production is plotted in Fig. 7. The concentration of product ran through a maximum value in each case and

608 100 • z.O _.= ~

15

80-

• 0,3

-30 60"

E

.o

o

20

9

40-

¸ 0,2 ~

~0

~5

10

20"

i

s.

&1

01 0

2~ o~

~

~

~

~,

Io

o

20

40 60 Bacteria[ wet weight

80 [gill

100

,~ .90 "~

0 20

40

60

Sucrose concentration

~_~

tl.

~

80

100

[g/I]

Fig. 8. Effect of initial substrate concentration on 3-keto-sucrose formation (@) and maximum yield (©). The reaction conditions were similar to those described for Fig. 7

Fig. 6. Influence of biocatalyst concentration on the formation of 3-keto-sucrose ( ~ ) and the specific rate of product formation (r~). Incubation was performed in 500-ml erlenmeyer flasks filled with 100ml of 0.1 M phosphate buffer, pH 7.0, at 27°C. The sucrose concentration was 50 g/1

10

40

30"

30

2 20 # 9

20

o

~

0

6

~ o

4

t_

113' 0

o~

10

10

20

30

40

50

6b

Reaction time [hi

'?

2

1'0 Reaction time [h]

Fig. 7. Role of substrate concentration on the time course of 3keto-sucrose formation. The initial sucrose concentrations were 5g/1 ( ~ ) , 1 0 g / l ( ~ ) , 25g/1 (O), 5 0 g / l (m), 75g/1 (r~) and 100g/1 (A). Incubation conditions were similar to those described for Fig. 6. The biocatalyst concentration was 50 g / l

Fig. 9. Effect of aeration rate on yield and formation of 3-ketosucrose. The reaction conditions were. similar to those described in Table 2 except that the pH was constant at 7.0 and the aeration rate was varied: x , 0.0 vvm; O, 1.9 vvm; A, 3.8 vvm; @, 7.7 vvm; rn, 11.5 vvm

then decreased. At high substrate concentrations the maximum of 3-keto-sucrose formation was reached later than at low concentrations, but distinctly higher product concentrations were obtained with high substrate concentrations. Several effects may be responsible for these results: oxygen transfer limitation, enzyme inactivation, and subsequent reactions of the product formed. In Fig. 8 the same results are plotted in a different way. This figure shows the dependence of maximum product concentration and maximum yield from the substrate concentration, where a countercurrent trend of product formation and yield becomes evident. A reasonable compromise for the production of the conflicting aims of product yield and product concentration must obviously be found. The importance of aeration was previously referred to by Bernaerts and De Ley (1961). They pointed out

that an oxygen consumption rate of the cells up to 6 Ixmol/min per litre solution should be provided, corresponding to an aeration rate of 1.6 vvm. At this aeration rate a conversion of 20% sucrose into 3-keto-sucrose was obtained. The influence of aeration rate and time course of product formation have been studied in the reactors shown in Fig. 2. As the residence time of the relatively large bubbles was rather short (reactor volume height 12 cm), a very high aeration rate was chosen in comparison to the usual aeration of fermentations. The effect of different constant aeration rates on product formation and yield is shown in Fig. 9. In a control reactor without aeration (except oxygen supply through the fluid surface) no significant 3-keto-sucrose formation was observed. The yield was increased by strong aeration; however, not in a linear relationship to the aeration rate. The best yield was reached at the highest aer-

609 ation rate, but already at the lowest aeration rate a high amount of 3-keto-sucrose was detected. This effect was studied in detail by Matalla (1990).

Discussion In our studies with the cultures of A. tumefaciens we found that mainly Mn 2+ and Fe 2+ ions had a profound influence on the yield of 3-keto-sucrose and hence the activity of the corresponding dehydrogenase (Table 1). Kurowski et al. (1975) demonstrated that Mn 2÷ decreased the synthesis of cytochrome c:oxidoreductase in sucrose medium thus providing more unoxidized substrate for bacterial growth. This effect was confirmed by Janssens et al. (1983). Various carbon sources were suitable for cultivation except for fructose, which did not provide a relevant formation of 3-keto-sucrose with" resting cells. The maximum hexopyranoside cytochrome c: oxidoreductase activity of cells derived from fermentations occurred at the beginning o f the exponential growth phase when the cell mass concentration was rather low (Fig. 3). The time course of the substrate decreases and 3-keto-sugar production is comparable to that described for 3-ketomaltose formation by Tyler and Nakamura (1971). Kurowski and Darbyshire (1978) also found a maximum for 3-keto-sucrose formation and activity of glucoside 3-dehydrogenase at 18-20 h. Recent results (not shown) indicate that this disadvantage can be overcome by substrate fed batch and pO2-shift during the cultivation period. With resting cells ofA. tumefaciens the product yield was 60% at a sucrose concentration of 5 g/1 and decreased to 40% at concentrations about 20 g/1 in shaking cultures. In the reactors described above (Fig. 2) a product yield o f 49% was obtained with 25 g/l of sucrose under appropriate conditions (Table 2). In contrast to the findings of Tyler and Nakamura (1971) with maltose, an increase in product formation was observed with increasing substrate concentration, but the yield was best at the lowest substrate concentrations (Fig. 8). Yields were favourable when the p H during the oxidation reaction was maintained between 7.0 an 7.6 (Table 2), but, at p H levels higher than 7.0, high amounts of fructose appeared in the reaction mixture indicating instability o f 3-keto-sucrose at alkaline p H levels. Under all conditions the maximum yields obtained were transitory. It is very likely that hydrolytic reactions and polysaccharide formation are responsible for the product degradation, as an increase in resting cell mass was observed in oxidation reactions without nutrient supply. Consumption of 3-keto-sucrose by the organism cannot be excluded as Janssens et al. (1983) described utilization o f 3-keto-lactose by a strain of A. tumefaciens belonging to the genetic cluster 2. Oxygen had a marked influence on the production o f 3-keto-derivatives and was reported by Walter (1990) and Walter et al. (1991) for leucrose. From these findings it is obvious that there exists an optimal oxygen concentration for the production of 3-keto-leucrose at

rather low stationary oxygen concentrations in solution. This effect may be the result of the transfer o f electrons and hydrogen to oxygen by two enzymes with a very different Km for oxygen. Furthermore, the yield of 3keto-sucrose is affected by follow-up reactions o f the product, as already mentioned. Small changes in reaction parameters such as concentration of biocatalyst, substrate concentration, oxygen supply or p H can change the time of maximum product concentration. Consequently a control o f product formation is advisable in each case in order to obtain optimal product yields that are distinctly better than those reported earlier and that make 3-keto-sucrose feasible for practical application in chemical synthesis. Our studies were extended to other substrates that underwent partial oxidation more efficiently with a higher yield o f new 3-keto-derivatives (Buchholz et al. 1991). These substrates included leucrose, isomaltulose and the corresponding aminoglycosyl-hexitols (2-amino-glucopyranosyl-mannitol and -sorbitol) as well as glucopyranosyl-arabinonic acid, in the form of its potassium salt (Noll, personal communication). With these derivatives, starting material for products such as diamines, surface active agents or emulsifiers becomes conceivable (Kunz 1991).

Acknowledgement. We gratefully acknowledge funds from the BMFT (Federal Ministery for Research and Technology, Bonn), project-no. 319250A2.

References Beeumen J van, De Ley J (1968) Hexopyranoside cytochrome c: oxidoreductase from Agrobacterium tumefaciens. Eur J Biochem 6:330-343 Beeumen J van, De Ley J (1975) Hexopyranoside cytochrome c: oxidoreductase from Agrobacterium. Methods Enzymol 41 : 153-158 Bernaerts MJ, De Ley J (1958) 3-Ketoglycosides, new intermediates in the bacterial catabolism of disaccharides. Biochim Biophys Acta 30:661-662 Bernaerts MJ, De Ley J (1960) Microbial formation and preparation of 3-ketoglycosides from disaccharides. J Gen Microbiol 22:129-136 Bernaerts MJ, De Ley J (1961) An improved method for the preparation of 3-ketoglycosides. Antonie van Leeuwenhoek, J Microbiol Serol 27:247-256 Bernaerts MJ, De Ley J (1963) A biochemical test for crown gall bacteria. Nature 197:406-407 Buchholz K, Stoppok E, Matalla K, Reh KD, Jrrdening HJ (1991) Enzymatic sucrose modification and saccharide synthesis. In." Lichtenthaler FW (ed) Carbohydrates as organic raw materials. VCH, Weinheim, pp 155-168 Fukui S, Hayano K (1969) Micro methods for determination of 3-ketosucrose and 3-ketoglucose. Agric Biol Chem 33:10131017 Fukui S, Hochster RM (1963) Conversion of disaccharides to the corresponding glycoside-3-uloses by intact cells of Agrobacterium tumefaciens. Can J Biochem Biophys 41:2363-2371 Fukui S, Hochster RM (1964) Carbohydrate inhibitors of sucrose uptake by resting cells of Agrobacterium tumefaciens. Can J Biochem 42:1023-1031 Fukui S, Hochster RM (1965) On the active transport of sucrose

610 and of 3-keto-sucrose in Aorobacterium tumefaciens. Can J Biochem 43:1129-1141 Grebner EE, Feingold DS (1965) o-Aldohexopyranoside dehydrogenase of Agrobacterium tumefaciens. Biochem Biophys Res Commun 19:37-42 Hayano K, Fukui S (1967) Purification and properties of 3-ketosucrose-forming enzyme from the cells ofA#robacterium tumefaciens. J Biol Chem 242:3665-3672 Hayano K, Fukui S (1968) Biochemical conversion of cellobiose to 3-ketocellobiose. J Biochem 65:901-903 Janssens D, Kersters K, De Ley J (1983) The significance of 3ketolactose in the lactose catabolism by Aorobacterium. Can J Microbiol 29:1096-1103 Kunz M (1991) Sucrose-based hydrophilic building blocks as intermediates for the synthesis of surfactants and polymers. In: Lichtenthaler FW (ed) Carbohydrates as organic raw materials. VCH, Weinheim, pp 127-153

Kurowski WM, Fensom AH, Pirt SJ (1975) Factors influencing the formation and stability of o-glucoside 3-dehydrogenase activity in cultures of Agrobacterium tumefaciens. J Gen Microbiol 90:191-202 Kurowski WM, Darbyshire J (1978) The production of 3-ketosucrose by Agrobacterium turnefaciens in batch culture. J Appl Chem Biotechnol 28: 638-640 Matalla K (1990) Mikrobielle Oxidation von Disacchariden. Doctoral Thesis, Technical University, Braunschweig Tyler DD, Nakamura LK (1971) Conditions for production of 3ketomaltose from Agrobacterium tumefaciens. Appl Microbiol 21:175-180 Walter J (1990) Untersuchungen zur mikrobiellen Leucrose-Oxidation. Diploma Thesis, Technical University, Braunschweig Walter J, Stoppok E, Buchholz K (1991) Einflussgr/Sssen der biotechnologischen Oxidation des Disaccharids Leucrose. Chem Ing Techn 63:631-633