DK170307B1 - Thermostable cyclodextrin glycosyl transferase, the preparation and use thereof, and a micro-organism for producing it - Google Patents

Description

DK 170307 B1

This invention relates to a cyclodextrin glycosyl transferase, a process for its preparation, a microbial strain capable of preparing it and its use in starch flow and in cyclodextrin preparation.

Cyclodextrin glycosyltransferase (1,4-α-D-glucan 4-α- (1,4-α-D-glucano) -5 transferase, EC 2.4.119), hereinafter referred to as cyclodextrin glycosyltransferase or CGTase is an enzyme characterized by its ability to form cyclodextrins by cyclizing starch or starch hydrolyzate.

Thus, the enzymatic cyclization reactions form cyclodextrins (abbreviated as CD and also known as Schardinger dextrins), these being 1-ring molecules consisting of 6,7 or 8 α-D-glucopyranose units linked by α-1,4 bonds. . They are known as α-, 8 or γ-cyclodextrins depending on the number of glucose units which are 6, 7 or 8, respectively.

Cyclodextrins have so far typically been prepared by variations of the method described by E.B. Tilden and C.S. Hudson (J. American Chemical Society) 64: 1432 [1942]; this method involves treating the liquefied starch with a cyclodextrin glycosyltransferase (CGTase) enzyme from Bacillus macerans. All variations of this process have a number of disadvantages. First, the starch must be pretreated, e.g. with an α-amylase, to solubilize the starch since the CGTase is not sufficiently heat stable to be used above starch adhesive temperature. It is important that the starch is transferred to a relatively low DE (Dextrose equivalent) so that after performing the starch transfer, the agent used, usually an α-amylase, must be inactivated to obtain a good yield of cyclodextrin. Second, Bacillus macerans CGTase is not sufficiently stable to be used at elevated temperature, and consequently, the enzymatic cyclodextrinization is performed at about 25 ° C, where it is exposed to microbial infection. Third, conversion of starch to cyclodextrins (at 50 ° C, pH 7.0) with Bacillus macerans CGTase requires long reaction time before reasonable yields are obtained.

The known CGTase enzymes are produced by microorganisms such as Bacillus macerans, Bacillus circulans, Bacillus stearothermophilus, Bacillus 30 megaterium, Bacillus ohbensis, alkalophilic Bacillus sp., Micrococcus luteus, Micrococcus varians and Klebsiella phenumoniae. None of the CGTase enzymes that are 2 DK 170307 B1 M.

however, formed by these microorganisms are sufficiently stable above 60 ° C to be used to produce cyclodextrins at sufficiently high temperature to avoid the possibility of microbial infection.

Enzymatic flow of aqueous starch slurry is widely used in the first step of the conversion of starch to dextrose (glucose).

The starch industry has largely introduced the process of liquefaction according to the US

3921590. Typical conditions are jet boiling at 105 ° C for 5 minutes followed by 90 minutes residence at 95 ° C, at a starch concentration of 35% DS (dry matter).

R

by weight. The enzyme used in this process is TERMAMYL (product of 10 Novo Nordisk A / S), an α-amylase from Bacillus licheniformis. The flow is carried out at pH about 6.0, followed by suction with giucoamylase at a pH of. about 4.5-5.0.

Starch transfer enzymes capable of flowing at pH 4.5 have long been sought to avoid the need for the intermediate pH adjustment. α-amylase from Bacillus stearothermophilus has been proposed for this purpose, but data in this application show that it does not perform well at pH as low as 4.5. US 4,578,532 and US 4,613,570 disclose acid-resistant α-amylases from Clostridium, but data in the patents in question show that their stability at 100 ° C and above at pH 4.5 is insufficient.

It is an object of this invention to provide a cyclodextrin glycosyl transferase with sufficient heat stability to be used for CD production at 60 ° C and above where the risk of microbial infection is low and even to be used for starch flow above 90 ° C where the starch is fully disguised.

It is also an object of the invention to provide an enzyme capable of providing starch at pH 4.5 and a temperature above 100 ° C.

Other objects of the invention are to provide a process for producing the heat stable enzyme in question and a microbial strain capable of producing it. It is furthermore an object to provide a progress <? The method utilizes said enzyme to prepare CD at 60 ° C and above and a method utilizing said enzyme for starch flow at pH about 4.5-5.0.

3 DK 170307 B1

The inventors have isolated a number of thermophilic, obligate anaerobic strains producing CGTases with surprising heat stability.

Accordingly, in its first aspect, the invention provides a cyclodextrin glycosyl transferase (CGTase), characterized in that it is derived from a CGTase-forming strain of Thermoanaerobacter or Thermoanaerobium and has a temperature optimum measured at pH 5.0 of about 95 ° C; a pH optimum of about 5.0; and a residual activity after 40 minutes of incubation at 80 ° C and pH 5.0 of about 95% without the presence of starch and Ca ++.

Another aspect of the invention provides a process for preparing the cyclodextrin glycosyltransferase of the invention, characterized in that a CGTase-forming strain of Thermoanaerobacter or Thermoanaerobium is grown under anaerobic conditions, after which the CGTase is recovered from the fermentation medium.

A third aspect of the invention provides a process for producing cyclodextrin glycosyltransferase according to the invention, characterized in that a transformed host organism containing the relevant genetic information from Thermoana aerobacter or Thermoana aerobium is grown under aerobic conditions in a suitable nutrient medium and then extracted from the CGTase. fermentation medium.

A fourth aspect of the invention provides a biologically pure culture of a strain of Thermoana aerobacter or Thermoana aerobium for use in the above method, characterized in that the strain is ATCC 53627, or one of the strains NCIB 40053 to NCIB 40059 or a mutant thereof which is capable of forming cyclodextrin glycosyltransferase having a temperature optimum measured at pH 25 of about 95 ° C; a pH optimum of about 5.0; and a residual activity after 40 minutes of incubation at 80 ° C and pH 5.0 of about 95% without the presence of starch and Ca ++.

A fifth aspect of the invention provides a process for liquefying starch, characterized in that an aqueous starch slurry is subjected to enzymatic liquefaction in the presence of the CGTase of the invention at a pH in the range of from 4.0 to 5.5, preferably at a temperature above 100 ° C.

4 DK 170307 B1

A sixth aspect of the invention provides a process for the preparation of dextrose, characterized by subjecting an aqueous starch suspension to enzymatic flow in the presence of the CGTase of the invention at pH in the range of about 4.0 to 5.5 (preferably at a temperature above 500 ° C), after which the liquefied starch sediment in the presence of glucoamylase is assayed, substantially without an intermediate pH adjustment.

A seventh aspect of the invention provides a process for preparing cyclodextrin, characterized in that a treated starch solution is treated with the cyclodextrin glycosyltransferase of the invention at a temperature above 60 ° C and then a cyclodextrin product is recovered from the reaction mixture.

Finally, an eighth aspect of the invention provides a process for the preparation of cyclodextrin, characterized by treating an aqueous starch suspension with the cyclodextrin glycosyltransferase of the invention at a temperature in excess of ca. 100 ° C and at a pH in the range 4.0-5.5, preferably substantially without the addition of a calcium salt, and then keep the syrup formed at a temperature in the range 80 ° -90 ° C for no more than 28 hours. , whereby the syrup is in the range 20-30 DS in each part of said residence time, and then a cyclodextrin product is recovered from the reaction mixture.

The microorganisms of the invention are thermophilic, obligate anaerobic bacteria belonging to the genus Thermoanaerobacter (J. Wiegel and L.G. Ljungdahl,

Arch. Microbiol. [1981] 128: 343-348) or the genus Thermoana aerobium (J. G. Zeikus et al., Arch. Microbiol. 122, 41-48 [1979]). The taxonomy of these genera has not been established, and it is considered likely that the two genera should actually be classified as one and the same genus, since they are so similar that even an expert in the field cannot distinguish them.

Unlike known strains of Thermoana aerobacter and Thermoana aerobium, the microbial strains of the invention are characterized by * 30 the ability to form CGTase and by being non-motile. Some strains of the invention are also indole positive in contrast to known strains. The known DK 170307 B1 strains T. ethanolicus DSM 3389 and T. finii DSM 2246 have been found not to form thermostable CGTase.

Eight strains were isolated by the inventors and deposited by the American Type Culture Collection (ATCC) and The National Collections of Industrial Marine 5 Bacteria (NCIB) for patenting purposes under the terms of the Budapest Treaty as follows:

Deponerinasnr. Date of deposit Depositor's reference 1. ATCC 53,627 June 3, 1987 ANO-15-7-5A2-70 2. NCIB 40,053 October 6, 1988 ANO-16-7-2A-70 10 3. NCIB 40,054 October 6, 1988 ANO-16- 7-4A-70 Γ 4. NCIB 40,055 October 6, 1988 ANO-36-7-1 5. NCIB 40,056 October 6, 1988 ANO-38-7-1 6. NCIB 40,057 October 6, 1988 ANO-44-5- 1-55 7. NCIB 40,058 October 6, 1988 ANO-51-7-1-70 15 8. NCIB 40,059 October 6, 1988 ANO-55-7-1-70

Mutants of the above strains having substantially the same properties are also within the scope of the invention.

These 8 strains were all classified by NCIB, Scotland as Thermoanaerobacter sp. or Thermoanaerobium sp., as it was not possible to determine which of these two genera were involved. Other taxonomic data are given below.

3 6 DK 170307 B1

Stammenr. 12345678 incubation at "C 55 60 60 60 60 60 60 60 5 _—----------

Cell morphology (a) (b)

Gram ------- var.

Spores --------

Motility -------- 10

Colony morphology (c) (d) (d) (d) (d) Growth 30 ° C (e) 37 ° C ND ND + 50 ° C +

Viscous by KOH test + + + + + + + + '20 Growth in (+) + + + + - + glucose Ye Growth in TYG + ND ND + ND - - + 25

Catalase --------

Oxidase, Kovacs ------- P-W-S (f) 30

Chloramphenicol Sensitivity + Hemolysis on 35 horse blood days

Litmus milk +

reduction PNPG

40 H2S formation +

See notes (a) - (f) on next page 7 DK 170307 B1

Notes: (a) Granular bars of varying length, in chains.

(b) Regular spells, "granular" staining, single or in short chains.

(c) (Starch agar, 3 days): round, regular, whole, smooth, low (?), convex s (?), hazy, yellowish brown, 2.5 mm diameter (d) (RCM, 3 days): round, regular, whole, smooth, shiny, transparent, flat, white, 3 mm diameter.

(e) + (slow 7 days).

(f) Peptone water sugar: no acid or gas.

} 10 ND Not determined.

8 DK 170307 B1

API 20A Anaerobic test 24 hours 60 ° C

5 Tribal no. 12345678

Indole + ---

Urease NC NC - - - NC NC

10 Acid from:

Glucose ---

Mannitol ---

Lactose --- Sucrose + -

Maltose + - +

Salicin + -

Xylose +++ t Arabinose - + 20 Glycerol ---

Cellobiose ± - +

Mannose + - +

Melezitosis -

Raffinose --- Sorbitol ---

Rhamnose - +

Trehalose ± (+) +

Gelatinase * - ++

Aesculin 30 hydrolysis +++ NC No change after incubation ± Weak reaction * May be high temperature failure reaction 35 The CGTase enzyme can be prepared by culturing a microbial strain of the invention (e.g. ATCC 53,627) under anaerobic conditions in a medium containing maltodextrin as C source, yeast extract and mineral solutions. Optimum pH and temperature for CGTase formation are pH 7.0 and 67 ° C. The enzyme is secreted into the fermentation medium, which shows that it is an extracellular enzyme.

Alternatively, CGTase of the invention can be prepared by aerobically culturing a transformant containing the relevant genetic information. In general, this method of preparation will comprise the following steps: (a) providing a suitable recombinant DNA cloning vector comprising DNA sequences encoding functions that facilitate gene expression and a DNA sequence encoding the CGTase of a Thermo Anaerobacter Thermal anaerobic strain; (B) transforming a suitable host organism with the cloning vector of step (a); (c) culturing the transformed host organism under aerobic conditions in a suitable nutrient medium and then recovering the CGTase from the medium.

Examples of suitable host organisms are strains of Escherichia, two Streptomyces, Bacillus or Aspergillus, preferably a strain of E. coli, B. subtilis, B. licheniformis or A. oryzae.

The CGTase can be recovered by removing the cells from the fermentation medium and then concentrating the fermentation liquid, e.g. by ultrafiltration.

In order to characterize the CGTase, the crude preparation from ATCC 15 53,627 was purified for homogeneity by DEAE-Sepharose chromatography, Chromatofocusing®, and acarbose-Sepharose affinity chromatography. Three components designated I, II and III were purified. Only one CGTase component was found in the crude preparations of the other strains based on SDS-polyacrylamide gel electrophoresis.

The CGTases of the invention are characterized by a thermostability far superior to prior art enzymes. After incubation in 5% Lintner starch -0.1 M sodium acetate pH 5.0 (50 ppm Ca ++) - for 50 minutes at 95 ° C, CGTase of the invention (ATCC 53,627) retains almost 100% of its activity.

Figure 3 shows the residual activity of crude CGTase from ATCC 53,627 after 40 25 minutes incubation at various temperatures at pH 5.0 without the presence of substrate and Ca ++. As shown, it retains approx. 95% of its activity at 80 ° C under these conditions. For comparison, data for two previously known □ flow enzymes are also shown: α-amylase from Bacillus licheniformis (Termanyl) and α-amylase from Bacillus stearothermophilus.

Components I, II and III have similar thermostability as the crude CGTase from ATCC 53,627. In comparison, it is reported that Bacillus 1 ° DK 170307 B1 macerans CGTase is only stable at temperatures below 50 ° C and rapidly loses activity above 50 ° C (Stavn, A. and Granum, PE in "Carbohydrate Research", 75 [1979 ] 243).

The influence of temperature on CGTase activity was investigated. The CGTase from ATCC 53,627 has a temperature optimum of 95 ° C at pH 5.0 in 0.1M sodium acetate - 100 ppm Ca ++ (see Figure 1). This optimum is in contrast to the optimum for Bacillus macerans CGTase, which has been reported to 55 ° C at pH 6.0 (Stavn, A. and Granum, P.E. in "Carbohydrate Research", 75 [1979] 243).

The influence of pH on CGTase activity was investigated at 60 ° C. The pH optimum for CGTase from ATCC 53,627 is 5.0 with broad activity in the acidic region 10 when tested in a buffer system of citrate phosphate-0.5% Lintner starch-100 ppm Ca ++ (see Figure 2). This value corresponds to the pH optimum of 5.2-5.7 reported for Bacillus macerans CGTase (Stavn, A. and Granum, P.E. in "Carbohydrate Research", 75 [1979] 243).

The molecular weight of the CGTases determined by SDS polyacrylamide gel electrophoresis followed by a coating of 0.8% Lintner starch iodine Gelrite® at pH

6.0, 55 ° C were as follows: ATCC 53,627 I 117,000 Daltons ATCC 53,627 II 110,000- ATCC 53,627 III 108,000- NCIB 40,053 99,000 - NCIB 40,054 106,000- NCIB 40,055 104,000- NCIB 40,056 101,000- NCIB 40,057 126,000 - NCIB 40,058 210,000 - NCIB 40,059 154,000 -

These results show that the CGTas are all different. «

Isoelectric focusing with a LKB Ampholine PAG plate pH 3.5-9.5 followed by coating with 0.8% Lintner starch iodagar at pH 6.0, 55eC has shown that the isoelectric points of CGTase I, II and lil are 4.55, 4.50 and 4.50 respectively.

11 DK 170307 B1 NPLC mec * Aminex® HPX-42A (Bio-Rad) with refractive index detector showed that the reaction patterns formed by the degradation of Lintner starch with each CGTase from ATCC 53,627 were similar (see Figure 4). The three CGTases are therefore catalytically similar. The three peaks on the right appear after hydrolysis and have been shown by NMR 5 (from left to right) α, γ and B-cyclodextrin, respectively.

In Figure 7, the reaction pattern formed by CGTase according to the invention (ATCC 53,627) is compared with a known fluid transfer enzyme, namely α-amyase from Bacillus stearothermophilus.

Determination of% conversion to α, γ and B-cyclodextrin during the flow with CGTase from ATCC 53,627 at a standard dosage of 4.46

Phadebas U7G DS is shown below. The conditions were 35% DS corn starch at pH

4.5 with primary flow at 105 ° C for 14 minutes and secondary flow at 90 ° C for 4 hours. In addition,% conversion with CGTase (6.8 Phadebas U / g DS) 2+ with 5% DS Lintner starch - 0.1 M sodium acetate (50 ppm Ca) at pH 5.0 and 95 ° C is shown for 50 minutes. Upon transfer, equal amounts of α and γ-cyclodextrin are formed, but almost twice as much B-cyclodextrin. In the reaction with Lintner starch, twice as much B-cyclodextrin as γ-cyclodextrin is formed, but 3 times as much α-cyclodextrin as γ-cyclodextrin.

Reaction Cyclodextrin

α-CD γ-CD β-CD

Lintner starch 13.8 4.4 9.3 25

Transfer 3.8 3.8 6.4

A comparison of CGTase according to the invention with published data for a number of known CGTases is shown in the table below. A number of distinct differences 30 occur, especially temperature optimum and stability DK 170307 B1 _ ——— 3 <«ϋ Ό O - - O o · o in oo · vo o in 'O' π u ft hoo vo t ** ό «Η · g 0 οι n cm o ιιοο KPU ins in h ms 2 oowm ft ό λ 5 -η ε- <« ^ in η ^ § _ «__ <__ S η - << 3 8» oj co ο ο - 3 ο ο ιη ο ο ι-l «Η« 3 * Ο · f '' '0' Β to μ · * · οηι co ο t "S -πω ο tn co in sii vo do a co ft in in κ * (0 Eh '-' 'aw _β __ <__ »t»> - 2 in a - - □ SCO O Or' o 9 η (0 σι ο ou * o - a η ρ «ψνοο · vo m« vo n -ho n in i in

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The liquefaction of starch serves to partially depolymerize and solubilize starch so that it becomes susceptible to subsequent enzymatic suction, e.g. with glucoamylase. The industry has largely chosen to use the flow process according to US 3,921,590. Typical conditions are heating to 105 ° C, e.g., 5 by jet boiling, staying for 5 minutes at that temperature followed by a 90 minute stay at 95 ° C with B. licheniformis α-amylase at ca. pH 6. Since glucoamylase is used at a pH of about 4.0-5.5, it has been desired to flow at a pH in this range, α-amylase from B. stearothermophilus flows well at pH 5.8, but both -amylases are unable to flow below pH 5.0. The liquefaction process of this invention is carried out at about pH 4.0-5.5 (preferably 4.5-5.5) whereby subsequent suctioning can be carried out without intermediate pH adjustment.

The CGTase of this invention does not require Ca ++ for stability even at low pH, so addition of calcium salt is not usually necessary.

15 Suitable flow conditions are approx. 1 -60 minutes at approx. 100-115 ° C, preferably followed by residence for approx. 50 minutes to 4 hours at approx. 80-100 ° C. A continuous process is preferred and heating is preferably done by jet boiling. The CGTase of the invention liquefies starch well at dosage levels at 2-5 Phadebas U (see below) per day. grams of starch DS (dry matter). The starch concentration will usually be in the range of 15-40% DS (wt% dry matter), often 25-35% DS.

α-amylase catalyzed hydrolysis of starch results in a reduction of viscosity simultaneously with an increase in reducing sugar. CGTase also breaks down starch but largely without the formation of reducing sugar. The enzymatic reactions significantly decrease the degree of polymerization, thereby producing a solution containing high molecular weight maltodextrins together with a considerable content of α-, 8- and γ-cyclodextrins. Thus, the conversion of 35% DS corn starch at pH 4.5 from liquefaction at 105 ° C for 14 minutes and a residence period at 90 ° C of 4 hours to α-, 8- and γ-cyclodextrin are thus 3, 8%, 6, respectively. 4% and 3.6%.

The liquefaction process of the invention can be used to prepare high yield dextrose (glucose) from wet milled corn starch or other refined starch by liquefaction with the CGTase followed by sugution with glucoamylase alone or together with pullulanase.

14 DK 170307 B1

The liquefaction process of the invention may also be used to prepare ethanol from starch-containing biomass. In this case, the biomass is displaced with CGTase at a pH of 4.5-5.5 followed by sugution with glucoamylase to form glucose and simultaneous or subsequent fermentation of the glucose to ethanol with yeast.

Subsequently, the alcohol can be recovered by known methods. Preferably, the entire process is carried out at a pH of about 5.0 without any intermediate pH adjustment, and at the same time suctioning and fermentation is carried out at ca. 30 ° C for up to 72 hours. The offset can be performed either at low DS levels (DS 15-20%) or high DS levels (DS 20-40%). In the processes of high DS, the DS level must be reduced to approx. 20% prior to fermentation to achieve a maximum yeast tolerance of approx. 10% by volume of alcohol.

The raw material for alcohol production may include refined starch, such as wet milled corn starch; raw, unprocessed materials such as maize, wheat, rice, sorghum, cassava and potatoes (whose starch content may range from 15-80%); and other starch-containing materials, such as industrial waste and by-products. In the case of refined starch, the displacement process preferably comprises a pretreatment above 100 ° C followed by a stay at a lower temperature to complete the displacement. In the case of other raw materials (having a lower starch content), the flow is preferably carried out in the range 60-100 ° C.

This invention provides a process for the preparation of cyclodextrins with thermostable CGTase. In the process of this invention, the liquefied starch is treated enzymatically with the CGTase at a temperature above ca. 60 ° C, preferably for no more than 24 hours and then the cyclodextrins are recovered from the reaction mixture.

In a preferred embodiment, the process comprises liquefying a starch slurry with the CGTase enzyme at ca. pH 5.0 at a pretreatment temperature above approx. 100 ° C, followed by conversion of the liquefied starch to cyclodextrins by holding the liquefied starch at ca. 80-90 ° C for up to approx. 24 hours in a residence step, preferably without pH adjustment and preferably without re-dosing the enzyme.

Contrary to prior art processes, the process of this invention utilizes temperatures sufficiently high to avoid substantial risk of microbial infection, which is of course advantageous. Another advantage associated with this is that the enzymatic conversion takes place faster with the CGTase at the high conversion temperatures. Processing time of less than approx. Twenty-four hours on an already liquefied starch substrate is contemplated in the practice of this invention. In spite of the above advantages, conversion of already starched starch is not a particularly preferred embodiment. It is far more advantageous to begin the process 5 with raw starch, e.g. a starch slurry and use the CGTase to form the displaced starch therefrom.

The CGTase enzyme can be used to displace starch (i.e., form a pourable syrup from a starch slurry) in the pH range 4.0-5.5 without the presence of added calcium and under standard starch flow conditions (as described above 10). Using the CGTase to liquefy a starch slurry and then to convert the liquefied starch into cyclodextrins is a highly advantageous process and is the preferred embodiment.

During the transfer of starch with CGTase, starch is converted to cyclodextrins simultaneously. Thus, a considerable amount of cyclodextrins is obtained and is present in the liquefied starch before being enzymatically converted to cyclodextrins.

The achievable yield of cyclodextrins directly from the starch transfer process is generally considered to be insufficient. In addition, the yield of cyclodextrins is significantly increased by enzymatic conversion of the 20 liquefied starch with CGTase.

The treatment of the (jet) cooked starch at high temperature stays which is part of the standard starch flow process can be extended to approx. 24 hours, however, preferably at approx. 90 ° C to convert more of the liquefied starch to cyclodextrins, whereby some dilution of the liquefied starch may be carried out to a more optimal DS level during conversion if desired. If a pH adjustment is desired for optimal enzymatic conversion to cyclodextrins, it can be performed either at the beginning of the starch slurry or simultaneously with the dilution. However, the re-addition of more CGTase enzyme after starch transfer has been found to be unnecessary.

Due to the thermostability of the cyclodextrin glycosyl transferase of the invention, which enables its use at higher enzymatic conversion temperatures than with prior art enzymes (especially higher than with 16 DK 170307 B1

The Bacillus macerans enzyme, whose temperature limit is 50 ° C), allows the combined liquefaction and conversion process to be carried out without any substantial intermediate cooling of the syrup. In other words, because of this ability to form high temperature cyclodextrins, the long reaction times hitherto used can be avoided, and in the practice of this invention, reaction times of less than about 1 24 hours for the enzymatic conversion of the liquefied starch. The starch used in the practice of the invention may be of any vegetable origin, such as the glutinosus variety of maize, common corn, wheat, sorghum, potatoes, tapioca, rice or sago. In addition to unmodified forms of the starch species, modified forms may be formed by treating starch with enzymes, acids, alkalis, etc. also used as substrates. The cyclodextrin-forming reactions may be effected on starch at DS concentrations of 1 to 40% DS, but for maximum conversion efficiency, a solution of 20-30% DS is preferred. If desired, more concentrated starch slurries can be displaced (with standard conditions being 35% DS) and then diluted to 20-30% DS dextrin solutions for conversion to cyciodextrins.

To summarize the conditions for the whole raw starch process as a starting material, the CGTase of the invention can be used under a variety of conditions, including the rather harsh standard conditions for liquefaction of a 35% DS slurry, namely jet boiling at 105 ° C and 90 minutes residence at 95 ° C. The pH range 4.0-5.5 without the presence of Ca ++ followed by a longer stay above 55 ° C for up to 24 hours. For maximum conversion and / or minimal use of CGTase, both residence steps (which of course can be a single extended stay) should be carried out in the range of 80-90 ° C and the starch concentration should be in the range 20-30% DS (either in the starch slurry). at the beginning or through dilution of the liquefied starch.

To further summarize, the cyclodextrins can be formed from the liquefied starch by incubating the syrup with the CGTase of the invention in the temperature range 50-95 ° C, preferably 80-90 ° C by reacting for approx. 24 hours or 30 less in the pH range 4-9, preferably about pH 5.0. The cyclodextrin product can be recovered from the reaction solution as before. In addition, the recovered cyclodextrins can be fractionated into α, IB and γ-cyclodextrins of the prior art, e.g. by DK 170307 B17 the methods described by D. French et al in Journal of the American Chemical Society 71: 353 (1949).

Batchwise conversion of starch hydrolyzate to cyclodextrins with Bacillus macerans CGTase has so far often been carried out in the presence of a suitable complexing agent to shift the equilibrium towards product formation. It is desirable that the cyclodextrin clathrates be insoluble and therefore precipitate from solution in the reaction mixture. The complexed cyclodextrin can be recovered by filtration or centrifugation and the complexing agent can then be separated by known methods. Suitable complexes for cyclodextrin include cyclooctane, hexane, 1-10 butanol, 1-decanol, etc. A number of complexing agents are known which selectively complex with the α or B form (see US Patent No. 3,640,847). Specifically, cyclooctane is selective for B-cyclodextrin while 1-decanol is selective for α-cyclodextrin. In the practice of this invention, it is contemplated to perform conversion with CGTase in the presence of complexing agents.

The activity of starch dextrinization of the CGTase is measured with Pharmacia

Phadebas (R) Amylase Test at pH 6.0, 60 ° C by incubating 200 µl of the enzyme solution with 4.0 ml of 0.1 M sodium acetate (100 ppm Ca ++) and a Phadebas Tablet for 15 minutes. The reaction is quenched with 0.5 ml of 1.0 N HCl.

The sample solution is centrifuged for 2 minutes in an Eppendorf centrifuge 20 at room temperature and the absorbance of the supernatant is measured at 620 nm, whereby an absorbance value of 1.0-3.0 should be obtained. A standard curve using Bacillus licheniformis α-amylase as a standard was set, in which one Phadebas unit is defined as the amount of enzyme that will catalyze the hydrolysis of 1.0 μmol of Lintner starch glucoside bonds. per minute at 60 ° C, pH 6.0.

The yields of α-, B- and -γ-cyclodextrins were determined by BioRad

Aminex® Carbohydrate HPX-42a High Performance Liquid Chromatography. Two columns (300 x 7.8 mm) were used in tandem at 85 ° C with glass-distilled water as eluent at a flow rate of 0.6 ml / minute. Detection occurred at refractive conditions. Standard curves were set up with authentic samples of α, B and γ-30 cyclodextrins (Sigma Chemical Company, St. Louis, Missouri).

Figure 1 is a representation of relative activity against temperature for the CGTase from ATCC 53,627.

18 DK 170307 B1

Figure 2 is a representation of relative activity against the pH of the CGTase from ATCC

53,627.

Figure 3 is a view of the thermostability of CGTase from ATCC 53,627 as compared to Bacillus stearothermophilus α-amylase and Termamyl®; Figure 4 is the HPLC measurements showing the reaction pattern of CGTase I, II, III from ATCC 53,627;

Figure 5 is a view of viscosity versus time comparing starch flow with CGTase from ATCC 53,627, Termamyl® and Bacillus stearothermophilus α-amylase; Figure 6 shows a torque plot against the rotational speed measurements showing the variations in viscosity of liquefied starch solutions obtained by varying dosage levels of CGTase from ATCC 53,627;

Figure 7 is the HPLC measurements comparing the reaction pattern of the CGTase from ATCC 53,627 and the α-amylase and Bacillus stearothermophilus.

EXAMPLE 1

Preparation of CGTase by Anaerobic Culture

The strain ATCC 53,627 was grown in a pre-reduced liquid medium under argon at an initial pH of 7 comprising the following components in grams 20 per gram. l:

Maltrin M-100, 5.0; KH 2 PO 4, 2.0; f ^ HPO, 6.0; NaCl, 1.0; (NH 4) 2 SO 4, 2.5;

MgS04.7H20,0,5; CaCI2.2H20,0,05; yeast extract, 2.0; Na, 0.5; cysteine HCl, 0.5; resazurin (redox indicator), 2 ng; and trace metals 5.0 ml. The trace metal solution consisted of the following components in grams per gram. liter: FeCl3.6H20.5.40; ZnSO4.7H2O, 1.45; MnCI2.4H20,1,00; CuSO4.5H2O, 0.25; and H3B03, 0.10. The trace metal solution was converted to concentrated HCl acid to dissolve the salts.

The strain was incubated at 67 ° C without stirring for 40 hours. The maximum activity after 40 hours was 200 Phadebas U per day. liter of liquid. The culture liquid 19 was centrifuged, then filtered and finally concentrated to a volumetric activity of 30-50 Phadebas U per liter. ml using a Millipore Minitan System.

Purification of CGTase from ATCC 53,627 was obtained by successive steps of DEAE-Sepharose chromatography, Chromatofocusing and 5 acarbose-Sepharose affinity chromatography. DEAE-Sepharose chromatography was performed at pH 7.5 in 10 mM Tris-HCl (2.5 mM CaCl 3, 4 ° C using a linear NaCl gradient 80-200 mM). Chromatofocusing (Pharmacia) was performed at 4 ° C using a linear pH gradient from pH 7-5. Acarbose affinity chromatography was performed at pH 6.0, 4 ° C, and elution was obtained by a 10 0.1 M sodium acetate (100 ppm Ca ++) buffer wash. Three distinct CGTase components designated I, II and III were obtained by Chromatofocusing and purified to homogeneity by acarbose affinity chromatography. The relative amount of the three CGTases obtained after chromatofocusing was CGTase I - 20%, CGTase II -60% and CGTase III - 20%.

EXAMPLE 2

Preparation of CGTase by Aerobic Culture of a Transformed Host Organism Chromosomal DNA was isolated from cells of strains ATCC 53,627 as follows. Cells (3.1 g wet weight) were suspended in 4.5 ml of 25% sucrose - 50 mM Tris pH 8.0 - 40 mM EDTA. The suspension was treated with lysozyme (2 mg / ml) for 30 minutes on ice followed by 30 minutes at room temperature. Pronase (1 mg / ml) was added and the suspension was incubated at 37 ° C for 30 minutes. The lysate was extracted twice with 8 ml of phenol for 30 min after the addition of 3 ml of 10 mM Tris -1 mM EDTa pH 7.4 buffer. The aqueous phase was then extracted twice with 10 ml of chloroform. The DNA was precipitated by the addition of 0.45 ml of 3M sodium acetate -25 1 mM EDTA pH 7.0 and 2.75 ml of isopropanol and incubated on ice for 5 minutes. The DNA was pelletized by centrifugation and washed once with 70% ethanol. The pellet was dried for 10 minutes under vacuum and then redissolved in 15 ml of 10 mM Tris - 1 mM EDTA pH 7.4 buffer.

The DNA preparation was subjected to a cesium chloride gradient centrifugation at 15 ° C, 192,000 g for 40 hours. The chromosomal DNA band was collected, extracted with isopropanol saturated with cesium chloride three times and dialyzed against 10 mM Tris - 1 mM EDTA pH 7.4 buffer at 4 ° C. A total amount of 590 µg of 5 chromosomal DNA was recovered.

The DNA was partially digested with restriction enzyme EcoRI by incubating 200 µg with 33 units of EcoRI in 150 µl of 50 mM Tris pH 8.0 - 10 mM MgCl 2 - 100 mM NaCl for 12 minutes at 37 ° C. The partially degraded DNA was subjected to 10-40% sucrose gradient centrifugation at 135,000 g, 15 ° C for 16 hours. 180 µl fractions were collected and 5 µl aliquots were analyzed by 1% Agarose gel electrophoresis. Fractions of the order of 7 to 15 kb were pooled, dialyzed against 1110mM Tris - 1mm EDTA pH 7.4 buffer, precipitated with ethanol, r and redissolved in 100 μ110mM Tris - 1mM EDTA pH 7.4 buffer.

The vector pBR322 was digested with Eco RI by incubating 2.5 µg with 15 units of Eco RI in 125 µl of 50 mM Tris pH 8.0 -10 mM MgCl 2 - 100 mM NaCl for 2 hours at 37 ° C. Analysis of the DNA on an Agarose gel showed that the degradation was complete. The degraded vector was extracted twice with phenol, once with ether and precipitated with ethanol.

The vector was dissolved in 100 µl of 100mM Tris pH 8.0 -1mM MgCl2 and 20 dephosphorylated with 20 units of calf intestinal alkaline phosphatase at 37 ° C for 30 minutes. The dephosphorylated vector was extracted with phenol twice, extracted with chloroform twice, and precipitated with ethanol. The dephosphorylated pBR322 was dissolved in 50 µl of 10 mM Tris - 1 mM EDTA pH 7.4 buffer.

Degradation of degraded pBR322 and strain ATCC 53,627 DNA was performed by mixing 0.4 µg of the chromosomal DNA (7-15 kb) partially digested with EcoRI and 0.1 µg of pBR322 which was digested with Eco RI and dephosphorylated with 10 units of T4 DNA ligase in 20 µl of 50 mM Tris pH 7.4-10 mM MgCl 2 -20 mM DTT -1 mM ATP containing 5 µg BSA / ml and overnight incubation at 14 ° C.

30 Competent E. coli HB101 (ATCC 33,694) cells were prepared for transformation by the method of T. Maniatus, E.F. Fritsch, and J. Sambrook in "Molecular Cloning" - A Laboratory Manual, page 250.

21 DK 170307 B1

Half of the ligated DNA prepared as above was transformed into competent cells by E. coli HB101 according to the method of Maniatus et al. (Supra). The cells were grown overnight at 37 ° C on plates containing Luria-Bertani (LB) medium and tetracycline at a concentration of 15 µg / ml. The tetracycline-resistant colonies were then transferred onto starch plates containing 1% amylopectin-LB medium and tetracycline (15 µg / ml), and incubated overnight at 37 ° C. Formation of CGTase was terminated by subjecting the starch plates to iodine evaporation after the heat treatment at 70 ° C for 1 hour, where clearance zones were observed.

A CGTase positive transformant designated E. coli NV601 was recovered from over 5000 colonies. The strain was both ampicillin and tetracycline resistant. Recovery of the recombinant plasmid by standard alkaline cleavage methods r and retransformation of competent E. coli HB101 cells yielded CGTase-positive transformants.

Restriction mapping of the recombinant plasmid revealed that a 12.8 kb DNA fragment had been inserted into the EcoRI position of pBR322. Deletion analysis with the restriction enzyme BamHI showed that the gene encoding CGTase was located on a 6.0 kb BamHI-BamHI fragment.

The CGTase was prepared by growing E. coli NV601 in Luria-Bertani 20 medium containing 5µ2 tetracycline per ml. ml of medium at 37 ° C, 300 rpm for 24 hours. The cells were collected by centrifugation and then sonically degraded.

Characterization of the recombinant CGTase relative to the native (native) CGTase in terms of molecular weight (SDS-PAGE), isoelectric point, thermostability, pattern of activity, flow activity and cyclodextrin production showed that there was no difference between the enzymes. The recombinant CGTase cross-reacted with antibody raised against the original CGT axis (component II).

EXAMPLE 3 DK 170307 B1

Cvclodextrinfremstillina

A comparison was made between the cyciodextrin yields prepared with the CGTase of the invention (ATCC 53,627) and the CGTase from Bacillus macerans. Since the Bacillus macerans CGTase is unable to flow starch under normal jet boiling conditions, a pretreated starch, ie. lintner starch, used. The enzymes were reacted with 15% DS Lintner starch (plus 40 ppm Ca ++) at 50 ° C and 90 ° C, pH 5.0 for 24 hours. The dosage was 4.46 Phadebas U per day. grams of DS starch. The CGTase from Bacillus macerans was also reacted at pH 7.0 as above as a blank test.

r

Cyclodextrinudbytte

CGTase pH Temp. α-CD y-CD β-CD Total Total CD

(* C)%% CD,% g / 100 ml

Invention 5.0 50 14.9 7.0 15.3 37.2 5.6 5.0 90 14.9 6.6 14.6 36.1 5.4 Bacillus 5.0 50 10.2 5.7 13 , 8 29.7 4.5 macerans 5.0 90 0.3 0 0 0.3 0.1 7.0 50 10.1 5.4 14.5 30.0 4.5 7.0 90 0.5 0 0.5 1.0 0.2 --------

The results show that CGTase according to the invention provides excellent conversion at 50 ° C, which is optimal for the known B. macerans enzyme. The CGTase of the invention shows essentially the same high degree of conversion at 90 ° C, where it is observed that the known enzyme is nearly inactive. The CGTase of the invention produces α-6- and γ-cyclodextrin in the ratio (at 50 ° C) 0.74: 1.0: 0.41, i.e. relatively more α-CD than the B. macerans enzyme.

EXAMPLE 4 DK 170307 B1

Starch at different pH values 35% DS corn starch with or without 40 ppm Ca ++ was floated at 105 ° C for 14 minutes followed by 4 hours at 90 ° C. The enzyme dosage was 4.46 Phadebas U / g DS (60 ° C, pH 6.0). CGTase of the invention (ATCC 53,627) was compared to Termamyl® (B. licheniformis α-amylase obtainable from Novo Nordisk A / S) and S. stearothermophilus α-amylase (available as G-Zyme® G995 from Enzyme Bio Systems, Ltd.).

Dextrose equivalent (DE) was measured after liquefaction, and the starch was considered to be liquefied if the starch syrup after liquefaction was pourable (showing a significant reduction in viscosity).

Enzyme pH Ca ++ DE Fluidated 15 CGTase 4.5 + 0.51 Yes CGTase 4.5 - 0.44 Yes CGTase 5.0 + 0.73 Yes CGTase 5.0 - 0.69 Yes CGTase 5.5 + 1.10 Yes 20 CGTase 5.5 - 0.83 Yes BS Amylase 4.5 + Not Determined No BS Amylase 4.5 - Not Determined No BS Amylase 5.0 + 4.78 Yes * 25 BS Amylase 5.0 - Can not determined No BS Amylase 5.5 + 9.78 Yes BS Amylase 5.5 - 5.58 Yes BS Amylase 5.8 + 13.6 Yes 30 Thermamyl 6.2 + 14.4 Yes * Considered elapsed, but very viscous.

It is seen that good flow can be achieved with CGTase according to the invention even at pH as low as 4.5 without the addition of Ca ++, and essentially without the formation of reducing sugars.

EXAMPLE 5 DK 170307 B1

Starch confluence at various enzyme doses

35% DS corn starch was liquefied with CGTase (ATCC 53,627) at pH

4.5 without the addition of Ca ++. Enzyme doses of 0.223,0,446,0,892,2.23 and 4.46 Phadebas U / g DS were used. In comparison, Thermamyl was used at pH

6.2 and S. stearothermophilus amylase at pH 5.8, both at 4.46 U / g DS and in the presence of 40 ppm Ca ++. Transfer conditions were 14 minutes at 100 ° C or 105 ° C (as indicated below), followed by 4 hours at 90 ° C. After displacement, viscosity was measured at 60 ° C with a Haake Rotovisco RV12 viscometer 1 o with NV sensor system and M500 "measuring drive unit" at the respective pH values for flow. Results are shown below and are shown in FIG. 6th

r

Enzyme Dosage Added pH Primary Viscosity (CP) U / g DS ++ flow, - at 15 Ca + temp. speed 32 CGTase 0.223 - 4.5 100eC *) CGTase 0.446 - 4.5 100eC *) CGTase 0.892 - 4.5 105 * C 208 20 CGTase 2.23 - 4.5 105 ° C 66.9 CGTase 4.46 - 4.5 105 ° C 42.4

Thermamyl 4.46 40 ppm 6.2 105 ° C 41.6 BS amylase 4.46 40 ppm 5.8 105 ° C 34.1 25 *) Measurement was not possible at this rate

The results show that a dosage level of 2-5 U / g DS of CGTase is suitable.

EXAMPLE 6

The pre-dried starch sugars 30 The pre-floured starch samples of Example 4 were adjusted to pH 4.3 or 4.5 at 60 ° C and Dextrozyme® 150/50 added at a dosage of 1.2 l / h DS.

DK 170307 B1 25

Dextrozyme® is a mixture of glucoamylase from Aspergillus niger and pullulanase from Bacillus acidopullulyticus; this can be obtained from Novo Nordisk A / S. Assay was carried out for 48 hours at 60 ° C and dextrose was determined by Bio-Rad Aminex® HPX-87C HPLC.

5 -----

Enzyme pH at pH at about 1% Dextrose flow. CGTase 4.5 4.5 + 96.0 CGTase 4.5 4.5 - 96.0 CGTase 5.0 4.3 + 95.6 CGTase 5.0 4.3 - 95.5 CGTase 5.5 4.3 + 95.7 CGTase 5.5 4.3 - 95.8 15 »BS Amylase 4.5 4.3 + Cannot be determined BS Amylase 4.5 4.3 - Cannot be determined BS Amylase 5.0 4 , 3 + 96.0 BS Amylase 5.0 4.3 - Cannot be determined BS BS Amylase 5.5 4.3 + 95.8 BS Amylase 5.5 4.3 - 95.9 BS Amylase 5.8 4, 5 + 96.8

Thermamyl 6.2 4.5 + 96.4 -----

The results show good suction ability of all starch liquefied according to the invention. The ability to sweeten starch flowed at pH 4.5 shows a clear advantage of the method of the invention over known fluxes with α-amylase from B. licheniformis or B. stearothermophilus, since only a small or no pH setting before suckering is necessary in the case of starch flow according to the invention.

EXAMPLE 7

Preparation of cvclodextrin at various starch concentrations

The corn starch was varied from 15% to 40% DS. The slurries were first liquefied at pH 5.0 without the addition of calcium in a treatment for 14 minutes at 105 ° C followed by 4 hours at 90 ° C, where the CGTase (ATCC 53.627) was used at a dosage. of 4.46 Phadebas U per. grams of DS starch. The production of cyclodextrin was controlled after the standing period of the starch transfer process was continued so that the dextrin solution with enzyme was incubated for an additional 24 hours at pH 5.0 and 90 ° C. Cyclodextrin yields were determined by Bio-Rad 5 Aminex Carbohydrate HPX-42A HPLC. results:

Cyclodextrin yield% DS a-CD γ-CD β-CD Total CD Total CD 10%%% g / 100 ml 15 9.6 6.3 15.4 31.3 4.7 20 8.1 5.8 17, 6 31.5 6.3 25 7.2 5.9 15.3 28.4 7.1 15 30 6.7 4.6 12.6 23.9 7.2 35 5.0 4.2 11.0 20.2 7.1 40 6.9 4.4 11.1 22.4 9.0

The conclusion was that an initial starch concentration of approx. 20-30% was optimal for the preparation of cyclodextrins, based on Total% CD and g CD / 100 ml. A concentration of 25% DS was therefore selected for further examples. 6-Cyclodextrin was prepared primarily at all starch concentrations. The ratio of β: α: γ-cyclodextrins was 1.0: 0.47: 0.39 at 25% DS. The highest yield of cyclodextrin from 25% DS starch observed was approx. 30%.

Reproduction with the CGTase enzyme and extending the reaction time to 48 hours did not increase the yields.

EXAMPLE 8

Preparation of cyclodextrin at various pH values

The effect of pH on cyclodextrin production was determined by using a 25% DS corn starch slurry. The starch slurries were flown at the indicated pH values, but otherwise as in Example 7, and the starch slurries were then incubated for 24 hours at 90 ° C at the same pH values.

The results shown below show that pH 5.0 was optimum for the combined process of starch flow and cyclodextrin preparation.

5 -

Cyclodextrin yield pH α-CD γ-CD β-CD Total CD Total CD%%% g / 100 ml 10 ------ 4.0 2.5 1.0 2.0 5.5 1.4 4.5 5 , 0 4.3 8.4 17.7 4.4 5.0 7.4 5.5 16.7 29.6 7.4 6.0 8.0 5.3 14.9 28.2 7.1 15 7.0 8.4 5.0 12.9 26.3 6.6, 8.0 8.4 5.0 13.4 26.8 6.7 9.0 6.3 4.5 8.5 19.3 4.8 Example 9 20 Preparation of cvclodextrin at various temperatures

The effect of temperature on cyclodextrin production by CGTase according to the invention (ATCC 53,627) was investigated without the addition of calcium by performing the incubation step for 24 hours at pH 5.0 at various temperatures as indicated below. A 15% DS or 25% DS slurry of corn starch was first sputtered with CGTase under the conditions of Example 7 at a dose of 4.46 Phadebas U per day. grams of DS starch. results:

15% DS

Cyclodextrin Yield 30 ------

Temperature a-CD 7-CD β-CD Total CD Total CD

% C%%% g / 100 ml 50 8.1 5.6 15.4 29.0 4.4 35 80 8.6 6.1 18.7 33.4 5.0 90 9.6 6.3 15.4 31.3 4.7 95 7.3 5.1 8.5 20.9 3.1 28 DK 170307 B1

25% DS

Cyclodextrin yield 5 Temperature α-CD γ-CD β-CD Total CD Total CD eC%%%% g / 100 ml 50 7.1 4.8 12.1 24.0 6.0 70 7.2 5.5 13, 7 26.4 6.6 10 80 7.2 5.5 14.6 27.3 6.8 90 7.2 5.9 15.3 28.4 7.1 95 6.3 5.8 14.6 26.7 6.7

The results show that the optimum conversion temperature is 80-90 ° C, both at 15% DS and 25% DS (after incubation for 24 hours). Lowering the temperature to 50 ° C gave less yield.

EXAMPLE 10

Preparation of Cyclodextrin at High Temperatures with and without Enzyme Gene Dose The possibility that equilibrium had not been obtained was investigated at 90 ° C and 95 ° C by re-dosing the reaction mixtures of Example 9 using 15% DS starch with CGTase before incubation for 24 hours and allowing the reaction to continue for another 24 hours. The results (see below) show that at 90 ° C equilibrium was obtained. At 95 ° C, it was necessary to re-dose to obtain the same 25 yields of cyclodextrins, showing a small loss of enzyme activity during prolonged incubation at 95 ° C.

29 DK 170307 B1

Cyclodextrinudbytte

Temp, Gendo Time α-CD γ-CD β-CD Total CD Total CD 5 (eC) Seration (hours)%%%% g / 100 ml 90 - 24 9.6 6.3 15.4 31.3 4 , 7 90 + 24 9.5 6.3 16.2 32.0 4.8 90 - 48 10.1 6.3 14.0 30.4 4.6 10 90 + 48 9.1 5.9 16, 5 31.5 4.7 95 - 24 7.3 5.1 8.5 20.9 3.1 95 + 24 10.2 6.5 13.7 30.4 4.6 95 - 48 9.3 5 , 2 10.2 24.7 3.7 15 95 + 48 11.3 6.6 13.5 31.4 4.7> EXAMPLE 11

Preparation of cvclodextrin with and without the addition of calcium

The effect of calcium on cyclodextrin production was investigated. A 25% 20 DS corn starch slurry was liquefied with CGTase of the invention (ATCC 53,627) at pH 5.0 with and without the addition of 40 ppm calcium at a dose of 4.46 Phadebas U per day. grams of DS starch. The liquefied starch samples were then incubated for 24 hours at 90 ° C. The results (see below) show that the presence of calcium ion had no effect on the overall yield.

25 -

Cyclodextrinudbytte

Calcium a-CD γ-CD B-CD Total CD Total CD%%% g / 100 ml 30 ------ 6.0 6.0 15.1 27.1 6.8 + 6.2 5, EXAMPLE 12 DK 170307 B1 30

Preparation of cyclodextrin at various enzyme doses

The effect on cyclodextrin yield of varying CGTase dosages was determined using 25% DS corn starch. The dosage ranged from 2.23 to 6.69 Phadebas U per day. grams of DS starch. The starch was liquefied at pH 5.0 using the doses given below in the presence of calcium with the CGTase from ATCC 53,627. The liquefied starch species were then incubated for 24 or 48 hours at 90 ° C, pH 5.0. Other conditions were as in Example 5.

The results (see below) show that after 24 hours and 48 hours, the 10 highest yields were reached at doses of 4.46 and 3.35 Phadebas U per day. grams of DS starch.

cyclodextrin

Doses, Phadebas Time, α-CD γ-CD β-CD Total CD Total CD 15 u grams DS hours%%% g / 100 ml 2.23 24 5.8 5.9 13.0 24.7 6.2 3.35 24 6.3 5.8 14.9 27.0 6.8 4.46 24 6.0 6.0 15.1 27.1 6.8 20 6.69 24 5.9 5.2 10.5 21.6 5.4 2.23 48 6.7 6.7 14.8 28.2 7.1 3.35 48 6.7 6.5 15.3 28.5 7.1 4.46 48 6.7 6.1 15.8 28.6 7.1 25 6.69 48 6.6 5.6 13.7 25.9 6.5 EXAMPLE 13

Preparation of cvclodextrin using comole formers

The effect of cyclooctane as a β-cyclodextrin complexer in the conversion of cyclodextrins was investigated. A 25% DS corn starch slurry was floated at pH 5.0 without the addition of calcium with the CGTase from ATCC 53,627 under the conditions of Example 7 at a dose of 4.46 Phadebas U per day. grams of DS starch. Then a conversion to cyclodextrin at 90 ° C followed for 90 hours for 24 hours, but cyclooctane was added on the order of 0.6 grams per minute. grams of DS starch before 24 hours of incubation began.

The conversion results (see below) showed that the addition of cyclooctane increased the final cyclodextrin yield, especially with respect to 6-5 cyciodextrin.

Cyclodextrin yield α-CD γ-CD β-CD Total CD Total CD 10%%% g / 100 ml

Control 7.2 5.9 15.3 28.4 7.1

With Cyclooctane: Without Complex 3.7 2.3 2.6 8.6 2.2! formation

With complex formation 1.9 0.6 25.0 27.5 6.9 Total 5.6 2.9 27.6 36.1 9.1 EXAMPLE 14

The forwardillina of cvclodextrin from different starch species

A comparison of several starch species was made.

Twenty-five slurries (25% DS) of corn, potato, wheat, rice and common corn starch were floated at pH 5.0 without the addition of calcium with CGTase of the invention (ATCC 53,627) at a dose of 4.46 Phadebas U per day. grams of DS starch under the conditions of Example 7. The liquefied starch solutions were then incubated for 24 hours at 90 ° C.

The results (see below) showed that there was a difference in the final cyclodextrin yield and that β-cyclodextrin was the primary product in all cases.

32 DK 170307 B1

Starch Cyclodextrin yield α-CD 7 "CD β-CD Total CD Total CD 5%%% g / 100 ml

Corn 7.4 5.4 15.4 28.2 7.1

Potato 9.3 5.6 14.6 29.5 7.4

Wheat 7.0 4.9 13.7 25.6 6.4 10 Rice 5.1 4.3 8.3 17.7 4.4

Aim. corn 7.1 4.5 11.9 23.5 5.9 EXAMPLE 15

Ethylene starch of precursed starch A 31.5% DS slurry of wet milled corn starch was liquefied with CGTase according to the invention (ATCC 53,627) at pH 5.0 without the addition of calcium for 14 minutes at 105 ° C followed by 4 hours at 90 ° C. A blank test with Termamyl® was also performed as described, but at pH 6.2 in the presence of 40 ppm calcium. The enzyme dose in each case was 5.0 Phadebase units per grams of DS starch. At the end of the transfer, the hydrolyzed starch was diluted to 22.4% DS with a yeast nutrient mixture. The final concentration of the components in the nutrient mixture per liter was 4.0 g of yeast extract, 1.6 g of ammonium phosphate, 0.4 g of magnesium sulfate, 3.2 g of citric acid and 0.6 g of sodium citrate. The final pH was 5.2. AMG 200 L (Novo Nordisk Bioindustrials, Inc., Danbury, CT) 25 was added at a dose of 0.44% wt / wt based on the starch. Penicillin G and streptomycin sulfate were included in amounts of 200 µg / ml. The fermentation mixtures were incubated at 30 ° C, 300 rpm for 64 hours.

The production of ethanol was measured indirectly by carbon dioxide evolution, ie. weight loss as a function of time. The final ethanol yields were confirmed with 30 Bio-Rad Aminex HPX-42A High Performance Liquid Chromatography.

After 64 hours, the yield of ethanol based on carbon dioxide production was 87.3% and 89.7% for CGTase and Thermamyl liquefied starches, respectively. These yields were verified by Bio-Rad Aminex HPX-42A HPLC. The standard industrial yield is usually approx. 86-90%.

EXAMPLE 16 DK 170307 B1

Preparation of CGTase by anaerobic culture of NCIB 40.053 - 40.059

The strains NCIB 40,053 to 40,053 were grown as described in Example 1 except that the temperature during cultivation was 55 ° C.

5 The maximum activity level after approx. 40 hours incubation i

Phage base units per liter of liquid cautious as follows: NCIB 40,053: 10, NCIB 40,054: 53, NCIB 40,055: 26, NCIB 40,056: 22, NCIB 40,057: 27, NCIB 40,058: 78 and NCIB 40,059: 10. The culture fluids were centrifuged, then filtered and finally concentrated for a volumetric activity of 30-50 Phadebase units per ml using the Miilipore Minitans system.

EXAMPLE 17

Starch staining with CGTase from NCIB 40,053 - 40,059 The CGTase preparations are prepared as in Example 16 were compared for their ability to flow 35% DS corn starch at pH 4.5. The starch was liquefied at 105 ° C for 114 minutes followed by 4 hours at 90 ° C at an enzyme dose of 4.46 Phadebas U / g DS (60 ° C, pH 6.0).

Dextrose equivalent was measured after liquefaction and the starch was counted as liquefaction if the syrup after liquefaction was pourable, which indicated a significant reduction in viscosity.

The results showed that all the CGTases could achieve good flow at pH

4.5, like the CGTase from strain ATCC 53,627. In all cases, essentially no DE could be measured, indicating CGTase activity.

Aminex® HPX-42A (Bio-Rad) HPLC, using refractive index, showed that the reaction pattern formed by the transfer of corn starch was typical of 25 CGTase, with the three peaks being α-, γ- and 8-cyclodextrin. No significant difference was observed in the relative ratio of the cyclodextrins prepared with each enzyme compared to the CGTase from ATCC 53,627.

EXAMPLE 18 34 DK 170307 Pg

Confused with Cloned CGTase

A 35% corn starch slurry was treated with cloned CGTase prepared as in Example 2 at a dosage of 8.92 Phadebas units per g DS 5 at pH 4.5 without the addition of Ca ++. Jet boiling was performed at 105 ° C for 5 minutes (primary flow) followed by standing at 95 ° C for 2 hours or 90 ° C for 4 hours (secondary flow). During secondary flow at 95 ° C or 90 ° C, a rapid reduction in viscosity was observed. At 90 ° C, the viscosity reduction was measured with a measured time using a Nametre viscometer. The results showed that there was a rapid reduction in viscosity to 400 centipoise x g / cm3 7 minutes into the secondary flow. The reaction pattern of the liquefied starch species after secondary liquefaction determined by Bio-Rad Aminex HPX-42A HPLC showed the characteristic cyclodextrin pattern at both temperatures. DE values of <1.0 were obtained by the neocuproin method, which shows the absence of reducing end groups according to the mechanism of a CGTase.

EXAMPLE 19

The sugars of starch confluence with cloned CGTase

The starch solutions liquefied with CGTase at pH 4.5 in Example 18 were aspirated with AMG and Dextrozyme at pH 4.5, 60 ° C for 48 hours at a dose of 0.18 AG per day. gram DS. The dextrose yields were determined by Bio-Rad Aminex HPX-87C HPLC.

«Β 35 DK 170307 B1% Yield

Enzyme Dextrose DP2 DP3 DP4 + 5 ----- 95 ° C Secondary flow

Dextrozyme 95.87 2.44 0.39 1.30 AMG 95.09 2.27 0.36 2.28 10 90 ° C Secondary effect

Dextrozyme 95.37 3.34 0.40 0.89 AMG 95.36 3.21 0.38 1.05

The results show a good yield of dextrose in all cases. The greatest yield was obtained by secondary flow at 95 ° C and saccharification with Dextrozyme.

t

Claims (11)

Cyclodextrin glycosyltransferase (CGTase), characterized in that it is derived from a CGTase-forming strain of Thermoana aerobacter or Thermo-anaerobium and has a temperature optimum measured at pH 5.0 of about 95 ° C; 5 a pH optimum of about 5.0; and a residual activity after 40 minutes of incubation at 80 ° C and pH 5.0 of about 95% without the presence of starch and Ca ++. The cyclodextrin glycosyl transferase according to claim 1, characterized in that the strain is ATCC 53,627 or one of the strains NCIB 40,053 to NCIB 40,059. Process for producing the cyclodextrin glycosyltransferase 10 (CGTase) according to claim 1, characterized in that a CGTase-forming strain of Thermoanaerobacter or Thermoanaerobium is grown under anaerobic conditions and the CGTase is recovered from the fermentation medium. Method for producing the cyclodextrin glycosyl transferase (CGTase) according to claim 1, characterized in that a transformed host organism containing the relevant genetic information from Thermoanaerobacter or Thermoanaerobium is grown under aerobic conditions in a suitable nutrient medium and the CGTase is recovered from the fermentation medium. . Process according to claim 3, characterized in that the strain is ATCC 53,627, one of the strains NCIB 40,053 to NCIB 40,059 or a mutant thereof having substantially the same properties. Method according to claim 4, characterized in that the host organism is a strain of Escherichia, Streptomyces, Bacillus or Aspergillus, preferably a strain of E. coli, B. subtilis, B. licheniformis or A. oryzae. DK 170307 Bl Biologically pure culture of a strain of Thermoana aerobacter or Thermoana aerobium for use in the method of claim 3, characterized in that the strain is ATCC 53627, or one of the strains NCIB 40053 to NCIB 40059 or a mutant thereof capable of forming cyclodextrin glycosyltransferase having a temperature optimum measured at pH 5.0 of about 95 ° C; a pH optimum of about 5.0; and a residual activity after 40 minutes of incubation at 80 ° C and pH 5.0 of about 95% without the presence of starch and Ca ++. Process for starch liquefaction, characterized in that an aqueous starch slurry is subjected to enzymatic liquefaction in the presence 10 of the CGTase according to claim 1 or 2 at a pH in the range of approx. 4.0 to 5.5, preferably at a temperature above 100 ° C. Process for the preparation of dextrose, characterized in that an aqueous starch slurry is subjected to enzymatic flow in the presence of the CGTase according to claim 1 or 2 at a pH in the range of approx. 4.0 to 5.5 (preferably at a temperature above 100 ° C), after which the liquefied starch slurry in the presence of glucoamylase is suctioned substantially without an intermediate pH adjustment. Process for the preparation of cyclodextrin, characterized in that a pretreated starch solution is treated with the cyclodextrin glycosyl transferase according to claim 1 at a temperature above 60 ° C and then a cyclodextrin product is recovered from the reaction mixture. Process for the preparation of cyclodextrin, characterized in that aqueous starch slurry is treated with the cyclodextrin glycosyltransferase according to claim 1 at a temperature in excess of approx. 100 ° C and at a pH in the range of 4.0-5.5, preferably substantially without the addition of a calcium salt, and then the syrup formed at a temperature in the range of 80 ° -90 ° C is kept at no more. than 28 hours, whereby the syrup is in the range 20-30 DS in each part of said residence time, and then a cyclodextrin product is recovered from the reaction mixture.