Appl Microbiol Biotechnol (2002) 59:609–617 DOI 10.1007/s00253-002-1057-x

MINI-REVIEW

A. Biwer · G. Antranikian · E. Heinzle

Enzymatic production of cyclodextrins

Received: 26 February 2002 / Revised: 24 May 2002 / Accepted: 27 May 2002 / Published online: 16 July 2002 © Springer-Verlag 2002

Abstract Cyclodextrins (CD) are enzymatically modified starches with a wide range of applications in food, pharmaceutical and chemical industries, agriculture and environmental engineering. They are produced from starch via enzymatic conversion using cyclodextrin glycosyl transferases (CGTases) and partly α-amylases. Due to its low solubility in water, separation and purification of β-CD is relatively easy compared to α- and γ-CD. In recent years more economic processes for γ-CD and especially α-CD production have been developed using improved CGTases and downstream processing. New purification steps, e.g. affinity adsorption, may reduce the use of complexing agents. The implementation of thermostable CGTases can simplify the production process and increase the selectivity of the reaction. A tabular overview of α-CD production processes is presented.

Introduction Cyclodextrins (CD) are cyclic oligosaccharides composed of α-1.4-glycosidic-linked glucosyl residues produced from starch or starch derivates using cyclodextrin glycosyl transferase (CGTase). There are three different types of CDs according to the number of glucosyl residues in the molecule: α-, β- and γ-CDs consist of 6, 7 or 8 glucose units respectively (see Fig. 1). Their water solubility differs significantly: α- and γ-CDs have a relatively high solubility (145 and 232 g/l), whereas the β-type is much less soluble in water (18.5 g/l). Further information on the physical, chemical and biological properties of CDs is given by Szejtli (1996a). A. Biwer · E. Heinzle (✉) Department of Biochemical Engineering, Saarland University, P.O. BOX 15 11 50, 66041 Saarbruecken, Germany e-mail: [email protected] Tel.: +49-681-3022905, Fax: +49-681-3024572 G. Antranikian Technische Mikrobiologie, Technische Universität Hamburg-Harburg, Kasernenstrasse 12, 21073 Hamburg, Germany

CDs are normally retailed as a dry, fine and crystalline powder, which remains stable long term. Due to its easier purification, the price of β-CD has decreased significantly in the past, whereas α- and γ-CDs are still more expensive. For industrial application, β-CD costs around US$ 3–4/kg, α-CD US$ 20–25/kg and γ-CD US$ 80–100/kg. In 1998, global consumption was around 6,000 metric tons, with an annual growth rate of 15–20% (McCoy 1999). Most CD sold is low-priced βCD but, with their prices coming down, market shares of α- and γ-CD are expected to increase significantly in the next decade. The main producers are Cerestar and Roquette Freres (France), Wacker Biochem (Germany) and Ensuiko Sugar and Nihon Shokuhin Kako in Japan (McCoy 1999).

CGTase There are a number of enzymes that can convert starch to various products of industrial value (Sunna et al. 1996). The enzyme catalysing CD production is CGTase [1,4-α-D-glucan 4-α-D-(1,4-α-D-glucano)-transferase, EC 2.4.1.19]. The enzyme produces non-reducing cyclic dextrins from starch, amylose, and other polysaccharides by catalysing different transglycosylation steps (Kobayashi 1996): intermolecular coupling und disproportionation, reacting at the α(1-4)-positions of oligosaccharides, and modification of the length of non-cyclic dextrins. Intramolecular cyclisation then leads to CD. Similar to amylases, CGTase can also hydrolyse starch. The molecular weight of CGTases varies between 60 and 110 kDa. Their properties depend heavily on the microorganism from which they are extracted (Prowe 1996; Starnes 2001). Today, many CGTases have been genetically improved. The enzyme is generally found in bacteria and was recently also discovered in archaea. Table 1 shows bacterial species producing CGTase which can be used for CD production. An extensive overview of such organisms and the properties of their enzymes is given by Kobayashi

610 Fig. 1 Chemical structure of α-, β- and γ-cyclodextrin (CD), consisting of 6, 7 or 8 glucose units respectively

Table 1 Bacteria species producing cyclodextrin glycosyl transferases (CGTase) for use in cyclodextrin (CD) production CGTase isolated from:

Reference

Bacillus macerans B. megaterium B. circulans B. stearothermophilus B. ohbensis B. licheniformes B. cereus Klebsiella pneumoniae K. oxytoca Micrococcus lutens M. varians Clostridium spp. Thermoanaerobacter spp. Thermoanaerobacterium thermosulfurigenes Anaerobranca gottschalkii

Shiraishi et al. 1989; Lee et al. 1992; Shieh and Hedges 1994; Starnes 2001 Bender 1986; Ammeraal 1988; Lee et al. 1992; Drauz and Waldmann 1995; Starnes 2001 Bender 1986; Mattsson et al. 1991; Lee et al. 1992; Van der Veen et al. 2000 Bender 1986; Ammeraal 1988; Lee et al. 1992; Drauz and Waldmann 1995; Starnes 2001 Bender 1986; Ammeraal 1988; Lee et al. 1992; Starnes 2001 Kobayashi 1996 Jamuna et al. 1993 Bender 1986; Ammeraal 1988; Lee et al. 1992; Drauz and Waldmann 1995; Starnes 2001 Lee et al. 1992; Choi et al. 1996 Yagi et al. 1982; Bender 1986; Lee et al. 1992 Yagi et al. 1982; Bender 1986; Lee et al. 1992; Starnes 2001) Slominska and Sobkowiak 1997 Lee et al. 1992; Starnes 2001 Wind et al. 1995, 1998; Starnes 2001 Prowe and Antranikian 2001

(1996). CGTases of Bacillus macerans have the biggest market share of the commercially available CGTases (Shieh und Hedges 1994). α-CD-producing CGTases are particularly known from Bacillus macerans, B. stearothermophilus and Klebsiella oxytoca (Bender 1986; Kobayashi 1996). In industrial CD production, CGTases from alcaliphilic Bacillus species are most commonly used (Yang und Su 1990). The production of CGTase is very similar to other enzyme production processes. Detailed information about the process can be found in, for example, Bender (1986), Gawande and Patkar (2001), Choi et al. (1996) and Lee et al. (1992). Heterologous production covers around 50% of the market. It is expected that this percentage will reach almost 100% in the next decade.

ture and environmental protection. Table 2 gives a short overview of possible and existing applications.

Production processes In general, two different types of CD production processes can be distinguished: In “Solvent Processes” an organic complexing agent precipitates one type of CD selectively and as such directs the enzyme reaction to produce mainly this type of CD. In the “Non-Solvent Process” no complexing agent is added and therefore a mixture of different CDs is formed. The ratio of CDs produced depends only on the CGTase used and on the reaction conditions. Efficient production processes using whole cell biotransformation are not known so far.

Applications Solvent process CDs have a cylindrical shape with a hydrophobic inside and a hydrophilic outside. Thus they are able to form inclusion bodies with many hydrophobic molecules, changing their physical and chemical properties. These and other properties make CD attractive for various applications in many different fields: food, chemical, pharmaceutical and textile industries as well as in biotechnology, agricul-

Figure 2a shows a typical flowsheet of a solvent process. Process data is taken from Schmid (1996), Ammeraal (1988), Bender (1986) and Hedges (1992). On an industrial scale, most CD is produced in solvent processes, where an organic solvent – mainly toluene, ethyl alcohol or acetone – acts as a complexing agent. The procedure

611 Table 2 Applications of CD within different industries Application

Industry

Reference

Stabilisation of volatile or unstable compounds

Food, pharmaceutical

Reduction of unwanted tastes and odour

Food, pharmaceutical

Gelling and thickening agent Protection from decomposition induced by light, temperature and air Removal of cholesterol Dietary fibre and calorie substitute for weight control Perfuming fabrics or loading with antiseptic substances Auxiliary material in production processes (e.g. fatty acids, benzyl alcohols, antibiotics) Improvement of bioavailability and reduction of side effects (e.g. Ibuprofen) Intermediate in drug production Separation of isomers Solution of water-insoluble compounds

Food Food Food Food

Horikoshi et al. 1981; Bender 1986; Hedges 1992; Maggi et al. 1998; Liese et al. 2000 Bender 1986; Hashimoto 1996; Nagai and Ueda 1996; Liese et al. 2000; Buschmann et al. 2001 Hashimoto 1996 Hedges 1992; Pedersen et al. 1995; Hashimoto 1996; Buschmann et al. 2001 Buschmann et al. 2001 Lee et al. 1992

Textile

Denter et al. 1997; McCoy 1999

Biotechnology

Bar 1996

Pharmaceutical

Additives in pesticides Control of plant growth Immobilisation of toxic compounds (e.g. heavy metals, trichloroethene) Improving decomposition of stable compounds (e.g. trichlorfon) and sewage sludge

Agriculture Agriculture Environmental protection

Nagai and Ueda 1996; Uekama and Irie 1996; Brunet et al. 1998; Maggi et al. 1998 McCoy 1999 Hedges 1992; Szejtli 1996b; Brunet et al. 1998 Bender 1986; Hedges 1992; Pedersen et al. 1995; Hashimoto 1996; Buschmann et al. 2001 Szente and Szejtli 1996 Brunet et al. 1998 Bar 1996; Szejtli 1996b; Wilson 1999

Environmental protection

Szejtli 1996b

Fig. 2 a Solvent process for cyclodextrin production. b Non-solvent process for CD production (here for β-CD production) (Schmid 1996)

Pharmaceutical Chemistry Chemistry, food

612

starts with starch liquefaction (typical starch concentration 20–30%). The liquefaction is carried out using either heat-stable α-amylase, acids (e.g. HCl), mechanical disintegration or thermostable CGTases. Additionally, some α-amylases require calcium ions (Seres et al. 1989). If α-amylase is used, it has to be inactivated by heat before further processing. On an industrial scale, liquefaction is normally achieved by jet-cooking. After liquefaction, the starch solution is cooled down to the enzyme reaction temperature, and CGTase and organic complexing agent are added. CDs are enzymatically produced and the desired type of CD forms a complex with the complexing agent and precipitates. After the conversion stops, the CD-agent complex is separated from the reaction solution by centrifugation or filtration. The remaining solution contains unused starch, linear dextrins, glucose, maltose, CGTase, unused organic complexing agent, some other by-products and water. The separated complex is washed, and the filtrate is distilled to recover excess complexing agent. In the next step, the CD-complex is suspended in water and cleaved by heating. The complexing agent is then separated by steam distillation. Some complexing agents cannot be isolated by distillation and have to be extracted by liquid-liquid extraction. The product solution is concentrated via vacuum distillation; sometimes the solution has to be treated with activated carbon. In the crystallising step, CD is precipitated, then filtered, washed and dried. The downstream process only separates the CDs from the rest of the reaction solution, but does not separate different CDs from each other. Thus, the choice of an appropriate enzyme and complexing agent determines the selectivity of CD formation and recovery. Non-solvent process Figure 2b shows a flowsheet of a typical non-solvent process. Process data is taken from Schmid (1996), Yang and Su (1990), Horikoshi and Nakamura (1979) and Hedges (1992). The non-solvent process was first developed for β-CD production. At an industrial scale, it is particularly used in Japan. Due to its low solubility, βCD can be easily purified by crystallisation steps. Purification of α- and γ-CD was achieved via complex and expensive chromatography with low yields and a range of by-products. However, more economic processes have recently been published, which also may have some potential environmental advantages. CDs produced without an organic complexing agent can be applied in the food industry without restriction, in contrast to CDs produced in solvent processes. β-CD production starts with starch liquefaction and enzymatic conversion identical to that used in solvent process, but with the exception that no complexing agent is used. At the end of the reaction, CGTase is inactivated, the pH is reduced and glucoamylase is added. The glucoamylase converts unused starch and other non-cyclic dextrins, which may disturb purification, to glucose

and maltose. The solution is then cleared using activated carbon, filtered and concentrated under reduced pressure to about 60% (w/v) dissolved solids. In some cases deionisation is necessary before the activated carbon treatment. After crystallisation and recrystallisation, the precipitated β-CD is isolated, washed, centrifuged and dried. The rest, consisting of glucose, maltose, α- and γCD, is concentrated to a syrup and can be used as a food additive. Compared to the solvent process, Schmid (1996) specified a lower yield of enzymatic reaction, a more complex purification process including an ineffective crystallisation step, a higher energy demand and a large number of by-products as the major disadvantages of the classical non-solvent process. Enzymatic conversion In general, the type and amount of CDs formed depends on substrate, origin of the CGTase, complexing agent and reaction conditions (Blackwood and Bucke 2000). Yield and selectivity are crucial. Therefore, new CGTases are sought or known enzymes are genetically improved to increase yield and selectivity. For instance, Van der Veen et al. (2000) achieved a 2-fold increase in the production of α-CD from starch using site-directed mutagenesis of a CGTase from Bacillus circulans. Wind et al. (1998) improved selectivity of a CGTase from Thermoanaerobacterium thermosulfurigenes using a combination of X-ray crystallography and site-directed mutagenesis. The additional use of debranching enzymes, e.g. pullulanases and isoamylases, added before the actual enzymatic CD production, can increase the yield by 4–6% (Schmid 1996; Rendleman 1997; Grull and Stifter 2001). Lima et al. (1998) published a production process for β-CD, combining an enzymatic CGTase reaction with yeast fermentation. The yeast consumes inhibitory compounds produced by the enzymatic conversion, e.g. glucose or maltose, thus increasing the CD yield. Rozell (1990) and Rohrbach and Scherl (1989) patented an immobilisation process for CGTases. Further information about immobilisation is also given by Bender (1986) and Yang and Su (1989). Tardioli et al. (2000) introduced an effective production process for βCD in a fluidised-bed reactor with immobilised CGTase. Yield and selectivity depend on type of CGTase and on the different substrates. Starch is the starting point for CD synthesis. Starch consists of amylopectin and amylose. Both can serve as raw materials for CD formation, but amylopectin gives higher yields than amylose. Therefore, it is difficult to transfer results from laboratory-scale processes with pure amylopectin to industrialscale processes, because the yields with normal starch are significantly lower and pure amylopectin is too expensive to be used in industrial processes (Grull and Stifter 2001). For high yield and trouble-free purification, the amount of impurities has to be small. For indus-

613

Fig. 3 Reaction scheme for CD formation using a complexing agent

trial production, potato starch is preferred. Corn and wheat starch contain a higher percentage of amylose and more impurities. Tapioca starch and waxy corn starch consist of almost 100% amylopectin and would be ideal substrates. However, these starches are currently too expensive and/or not available in sufficient quality and quantity (Schmid 1996; Slominska and Sobkowiak 1997; Grull and Stifter 2001). Besides using starch as raw material, some processes have been developed that use rice protein concentrate together with sugar syrup as a starting substance (Abelian et al. 1993). Improvement of raw material quality by genetic engineering of starch-producing plants is also being attempted. The development of an “amylopectin potato” with 90–98% amylopectin, inhibited amylose formation and a very low impurity content, could increase the yield of CD formation and simplify purification (Grull and Stifter 2001). A completely new way producing CDs is “Molecular Farming”. CDs are synthesised by genetically modified potato plants and extracted after the harvest. However, this research field is still in its infancy and so far yields are low. An economic production scale process within the next few years is quite unlikely (Schmid 1996). It has been known since the early twentieth century that the addition of organic complexing agents influences both yield and selectivity of CD formation. Complexing agent (complexant) and CD (host) form a complex that is insoluble in water and therefore precipitates (Fig. 3). The accumulation of α-CD during the enzymatic reaction inhibits its own synthesis and favours the formation of other CDs. This is similar in the case of β- and γ-CDs. The precipitation with a complexing agent reduces CD concentration and consequently product inhibition. An ideal complexant forms a complex selectively with only one type of CD. An overview of possible complexing agents in given by Rendleman (1997). Organic solvents like toluene, ethanol, butanol or propanol are frequently used as complexing agents. The addition of ndecanol, tetrahydrofurane, acetonitrile, 1-butanol, cyclohexane, aliphatic (acyclic) ester and various other C1–8aliphatic alcohols increases the α-CD yield. Toluene and a range of other compounds increase β-CD production, while bromobenzene and others favour production of γCD. The selectivity of the complexant depends also on the CGTase used (Flaschel et al. 1984; Blackwood and Bucke 2000; Starnes 2001). There are conflicting reports concerning the use of ethyl alcohol as a complexing

agent. Whereas Shiraishi et al. (1989) and Lee and Kim (1992) found a significant improvement in α-CD production, Mattsson et al. (1991) detected a slight reduction of α-CD formation and increased β-CD formation. In recent years, several processes using thermostable CGTases from extremophiles have been reported (Lee et al. 1992; Prowe 1996; Kim et al. 1997; Slominska and Sobkowiak 1997; Brunet et al. 1998; Lima 1998; Wind et al. 1998; Blackwood and Bucke 2000; Starnes 2001). Extremophiles are unique microorganisms that are adapted to survive in ecological niches such as high or low temperatures, extremes of pH, high salt concentrations or high pressure. Various enzymes from thermophiles (growth above 60°C) have been purified, and their genes cloned and expressed in mesophilic hosts, e.g. Escherichia coli and Bacillus subtilis. As a general rule, they show extraordinary heat stability, and are resistant to chemical reagents, detergents, urea and guanidinium hydrochloride. Optimal reaction temperatures with thermostable CGTases are between 60 and 90°C and are higher than in conventional processes where typical temperatures are around 20–40°C. Benefits in performing processes at higher temperature include reduced risk of contamination, improved reaction rate of enzymatic conversion, lower viscosity and higher solubility of substrates. Additionally, starch solution has to be cooled down to a lesser extent after the jet-cooking step (Slominska and Sobkowiak 1997; Starnes 2001). Furthermore, such CGTases can often be used for starch liquefaction. All CGTases can catalyse starch hydrolysis, but normal CGTases are not stable at the gelatinisation temperature of starch (around 90°C). Using thermostable CGTases eliminates the need for other enzymes like amylases. Thermostable CGTases are produced by, for instance, the anaerobic bacteria Thermoanaerobacter thermosulfurigenes and Anaerobranca gottschalkii (Prowe and Antranikian 2001). It is essential, however, to ensure overexpression of these enzymes in mesophilic hosts such as Bacillus sp., yeast (Pichia sp.), filamentous fungi or, more recently, Staphylococcus (see e.g. Sturmfels et al. 2001) and to optimise the cultivation process. Pedersen et al. (1995) described a process using CGTase of Thermoanaerobacter. Starch is first liquefied with CGTase at 105°C and then CD is produced in the next 4–24 h at 90°C without further addition of CGTase. In contrast to liquefaction with α-amylases, no oligosaccharides with low molecular weight are formed that could later disturb CD production and purification. Similar processes with consecutive or simultaneous starch liquefaction using thermostable CGTases have been published by Lee and Kim (1992), Slominska and Sobkowiak (1997), Yang and Su (1990) and Starnes (2001). Table 3 gives a general overview of processes with αCD as the main product. Normally α-CD production starts with starch, or starch hydrolysates, consisting of 1–20 dextrin equivalents (DE) and a maximum solid content of 50%. Usually, solid content is between 20 and 30%. The processes run at a pH of 4.5–8.5 and a temperature between 20 and 75°C. Process duration varies from

Thermo-anaerobacter spp.

Thermo-anaerobacter spp.

Clostridium thermo-amylolyticum

Clostridium spp.

Klebsiella oxytoca

K. oxytoca

K. oxytoca

K. pneumoniae AS-22

K. pneumoniae AS-22

K. pneumoniae M5al

Bacillus macerans

B. macerans

B. macerans

B. circulans 251

Micrococcus lutens and M. varians

Brunet et al. 1998

Kim et al. 1997

Starnes 2001

Slominska and Sobkowiak 1997

Lee et al. 1992; Choi et al. 1996

Schmid 1996

Schmid 1996

Gawande and Patkar 2001

Gawande and Patkar 2001

Flaschel et al. 1984

Shieh and Hedges 1994

Kim et al. 1995

Rendleman 1997

van der Veen et al. 2000

Yagi et al. 1982

10% (v/v) starch, CGTase: 6 U/g starch, 1.6 mM decan-1-ol, pullulanase: 0.45 U/ml 10% (w/v) potato starch (highly polymerised), CGTase: 0,1 U/ml 1% (w/v) starch, CGTase: 10 U/g starch, CA

25–30% (w/v)starch, ethyl alcohol, CGTase: 5 U/g starch 125 g/l wheat starch, 2% (v/v) butan-1-ol, 10 mM CaCl2 , CGTase: 20 U/g starch 500 g/l dextrin, 2% (v/v) hexan-1-ol, 10 mM CaCl2 , CGTase: 20 U/g starch 100 g/l starch, decanol: 0.1 l/kg starch, 5 mM CaCl2 30–35% waxen corn starch hydrolysate, 5% (v/v) cyclohexane, 600–700 T-H units/ml 7.5% starch, CGTase: 48 U/g starch

40–65°C, pH 4.5–5.5, 48 h

50°C, pH 6, 45–50 h

15°C, pH 7.2, 5 days

60°C, pH 6, 12–24 h

20–35°C, pH 6–6.5 1–4 days

40–50°C, pH 6.8, 6–24 h

40°C, pH 7.5, 6 h, flow rate UF: 600 l/h

40°C, pH 7.5, 6 h, flow rate UF: 600 l/h

40°C, 6 h

40°C, 4–24 (6) h

40°C, pH 6, 19 h

10% corn starch, 1 mM CaCl2 , CGTase: 10 U/g starch 25–30% (w/v) starch, decanol, CGTase: 5 U/g starch

90°C, pH 5, 5 h

80–85°C, pH 5–5.5, max. 24 h

65°C, pH 6, 24 h

90°C, pH 5

60°C, pH 6.1, 8 h

Reaction Conditions

5–25% potato starch, 0.5% CGTase

20–30% starch, CGTase thermostable

7.5% corn starch, CGTase: 22 U/g starch

10 mM maltose (different substrates tested), 0.1 mM β-CD 25% starch solution

Raw materials and additives 1. 95:4:1 2. 3. 1.8% α-CD 1. 3:5:2 2. 3. 1. 2. 12 mg/U CGTase 3. 27.9% 1. 8.1:17.6:5.8 2. 3. 31.5% 1. 51:32:17 2. 3. 25% α-CD 1. 96.5:3.5:0 2. 14.75 g/l 3. 1. 96.5:3.5:0 2. 3. 40–50% 1. 96.5:3.5:0 2. 3. 35% 1. 97:3:0 2. 3. 42.5% 1. 91:3:6 2. 55 g/l α-CD 3. 12.1% 1. 2. 3. 50% α-CD 1. 40:60:0 2. 107 g/l 3. 15.5% 1. 2. 5 mg/U CGTase 3. 25% 1. 75:0.8 :0.2 2. 3. 76% 1. 30:51:19 2. 3. 40% 1. 40:19:0 2. 3. 59%

Ratio and yielda

and yield: 1. α-CD:β-CD:γ-CD; 2. final CD concentration; 3. yield of CD with respect to starch used (total CDs unless otherwise stated)

Thermo-anaerobacter spp.

Brunet et al. 1998

a Ratio

Origin of the CGTase

Reference

GMO, optimized for industrial conditions

pullulanase as debranching enzyme (or isoamylase)

Starch: only short heat pretreatment

UF

With UF

Yields at industrial scale

Yields at industrial scale

no CA, no effect of higher substrate concentration

CaCl2 addition had no effect

CGTase also for starch liquefaction

starch not pretreated

thermostable MO

thermostable MO

Remarks

Table 3 Published CD production processes with α-CD as the main product considering raw materials, reaction conditions, yield and CGTase used. CA Complexing agent, T-H units Tilden-Hudson units, UF ultrafiltration unit, MO microorganism

614

615

10 h up to several days. Enzyme concentration has to be at least 45–90 U/g starch. A powerful stirrer is needed, because incomplete mixing significantly lowers the yield. Maximum yield is around 50% with respect to the starch added at the beginning (Ammeraal 1988; Shieh and Hedges 1994; Schmid 1996). Schmid (1996) describes industrial α-CD production using CGTase from Klebsiella oxytoca and decanol as a complexing agent. Decanol is needed in comparatively small amounts and can be recycled almost completely by steam distillation (Schmid 1996). In contrast, Pedersen et al. (1995) stated that α-CD cannot be produced at industrial scale with decanol as a complexing agent, the reason being the high boiling point of decanol (229°C), which could make its separation from the product difficult. Although widely used, the application of organic complexing agents in the solvent process has several disadvantages (Horikoshi et al. 1981; Okabe et al. 1993; Pedersen et al. 1995): the solvents are often toxic, are a potential environmental hazard, and are inflammable or have other safety risks. Furthermore, production is only economical if the solvent is nearly completely recovered and reused. This recycling incurs additional costs. The complete removal from the product is expensive limiting the use of the product in the pharmaceutical and food industries. Because of these disadvantages, other possibilities to overcome product inhibition and to improve purification are being sought. Most efforts have been made on the use of ion exchange chromatography, on synthetic adsorption materials and membrane filtration processes. These efforts have partly been linked with the switch from batch to continuous processes. They are described in the following section. Purification Using the typical solvent process purification procedure described above, it is possible to achieve purities of 98%. Most impurities are non-cyclic dextrins, the content of the organic complexing agent is often less than 1 ppm (Ammeraal 1988; Hedges 1992). However, solvent process purification only isolates the CDs from the rest of the reaction mixture, but not the different CD types from each other. The unwanted CD types, formed in the reaction and at least partly complexed, remain as impurities in the final product. Additional purification steps would be needed to isolate a single type of CD. Generally, three different processes for CD isolation and purification can be distinguished (Armbruster 1970; Tsuchiyama 1991b; Okabe et al. 1993): (1) precipitation with organic solvents (different from using complexing agents in the enzymatic reaction), (2) chromatography/adsorption (ion exchanger, affinity chromatography, molecular sieve, adsorption to synthetic polymers) or (3) membrane and filtration techniques. These methods can be used to partly replace the use of complexing agents. Due to the low water solubility, β-CD purification is relatively easy. In contrast, α- and γ-CD are much more

soluble in water. Therefore the separation of α- and γCD is much more demanding (Lee et al. 1992). According to Sato et al. (1994), chromatographical methods using ion exchangers or molecular sieves are most commonly used on an industrial scale. However, organic solvent extraction and, more recently, affinity adsorption are also implemented. Shieh and Hedges (1994) postulate a process for separating CDs based on the precipitation with an organic solvent. First, cyclohexane is used as a complexing agent in a standard solvent process and is then removed by steam distillation. A mix of α- and β-CD and some impurities remains in the product solution (γ-CD is not produced). After activated carbon treatment of the solution, β-CD is crystallised. Cyclohexane is then again added to the remaining solution and forms a complex with the remaining α-CD. The complex precipitates and can be separated from the impurities. The following downstream process is identical to the standard purification steps as described above and leads to highly pure α-CD. Yang and Su (1990) introduced a similar process where α-CD is isolated from the remaining reaction mixture of a nonsolvent process, as described above. After the removal of β-CD and the degradation of non-cyclic dextrins to short oligosaccharides by α-amylase or glucoamylase, α- and γ-CDs are precipitated with organic solvents and further processed as described above. Because of their low boiling point (easy distillation), trichloroethylene, dichloroethylene, chloroform and acetone have been used as organic solvents in this type of purification. Similarly, ion exchangers are used in industrial processes to separate α- and γ-CD from short oligosaccharides. The oligosaccharides are retained in a column, whereas CDs pass through (Horikoshi et al. 1981; Okabe et al. 1993). The yield of α-CD with respect to starch consumed is maximal at low substrate concentrations. Consequently, the overall α-CD concentration in the reaction mixture stays low because of the high dilution, which makes purification inefficient. However, with membranes using reversed osmosis the reaction mixture can be greatly concentrated at the end of the reaction. This simplifies the following purification steps significantly. Adsorption can be used after the enzymatic conversion as well as in integrated removal of CDs. CDs can be separated from by-products by adsorption to porous polymers (e.g. styrene bivinylbenzene copolymerisate). Afterwards, CDs are eluted and further purified in a crystallisation step. High yields can be achieved (Horikoshi et al. 1981; Japan Maize Products Co. 1981; Okabe et al. 1993). Affinity adsorption is applied especially to the purification of α-CD. Stearic acid, covalently bonded to chitosan beads, is used as a specific adsorbent. The selectivity of the adsorbent is 100%; no other CD type is retained. The actual adsorption capacity depends on the carrier material and its porosity, the ligand and its concentration, the temperature and the composition of the mobile phase. For purification to be economical, these parameters have to be optimised (Mäkelä et al. 1989;

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Tsuchiyama et al. 1991a; Okabe et al. 1993; Liese et al. 2000). Sato et al. (1994) and Nagano et al. (1987) have introduced a process where, at the end of the CD formation, CGTase and glucoamylase are again added to the reaction solution to convert the remaining starch and dextrins and also β- and γ-CD to α-CD and glucose (finally 30% α-CD and 70% glucose). α-CD is then isolated by affinity adsorption with a purification yield of 48.5%. A variety of processes have been published that keep the CD concentration low by continuous removal during the reaction. Thus the problem of product inhibition is solved and the use of complexing agents is not necessary (Hokse et al. 1984; Rohrbach and Scherl 1989; Tsuchiyama et al. 1991b; Kim et al. 1993; Okabe et al. 1993; Hong and Youm 2000; Liese et al. 2000; Gawande and Patkar 2001). However, some of these processes are economically inferior compared to conventional processes. Tsuchiyama et al. (1991b) and Okabe et al. (1993) described an economical α-CD production process with integrated removal of α-CD from the enzyme reactor by affinity adsorption. The reaction solution circulates through the reactor and the adsorption columns. α-CD is retained in the columns by stearic acid ligands. This keeps the concentration in the reactor low and reduces product inhibition. When the column is saturated, the concentration rises and the reaction stops. When the conversion has finished, the columns are washed, α-CD is eluted with hot water, concentrated, treated with activated carbon and crystallised. Purity of greater than 95% can be achieved. Relative to the starch used, an overall yield of 22% is reached. If α-CD is eluted without washing, a higher yield with a purity of 73% is possible, where the rest is β- and γ-CD. Liese et al. (2000) report that this process for α-CD production is used in Japan (Mercian). Besides adsorption, continuous removal can be performed by membrane or ultrafiltration units. In an integrated production process, an ultrafiltration unit can minimize α-CD concentration in the solution (Rohrbach and Scherl 1989). In industrial processes, ultrafiltration and reversed osmosis units are connected in series (Okabe et al. 1993). Many modified and improved procedures have been published based on the solvent and non-solvent processes described above. However, it is often difficult to assess to what extent research results are actually transferred to industrial production. In summary, an ideal combination of CGTase, raw material, reaction conditions and possibly a complexing agent is crucial to achieve an economic production process. Acknowledgements We thank The Deutsche Bundesstiftung Umwelt (DBU) for their financial support. We also thank Jens Kroemer for his help in compiling this review.

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