Method for producing alpha-cyclodextrin

Abstract

This specification provides a method for the enzymatic production of alpha-cyclodextrin from sucrose. Optionally, the method involves contacting sucrose with one or more enzymes to convert sucrose to amylose, and subsequently contacting amylose with one or more enzymes to convert amylose to alpha-cyclodextrin. Optionally, the method produces alpha-cyclodextrin in a higher yield compared to beta-cyclodextrin, gamma-cyclodextrin, or both.

Description

Technical Field

[0001] [Background Art] Cyclodextrin is a kind of cyclic oligosaccharide composed of cyclic oligomers of glucose. Cyclodextrin has a lipophilic core and a hydrophilic outer surface, and thus is useful in the pharmaceutical industry and various other industries. Natural cyclodextrins (i.e., α-cyclodextrin, β-cyclodextrin, and γ-cyclodextrin) have received Generally Recognized as Safe (GRAS) status from the US Food and Drug Administration (FDA) and are widely used, especially in the food and pharmaceutical industries. Standard methods for producing cyclodextrin generally involve the enzymatic conversion of starch. However, standard production methods have various disadvantages, including supply chain shortages, scalability, quality variations, and the cost of purification and goods. Therefore, there is a need for improved cyclodextrin production methods to address these problems.

Summary of the Invention

[0002] There is an unmet need for methods to produce cyclodextrin without using the conversion of starch as a starting material. The present disclosure meets this unmet need by providing a method for biosynthetically producing cyclodextrin without using starch as a starting material. The advantages of the present disclosure provided herein compared to other methods (e.g., starch-based methods) include, but are not limited to, an increase in the overall yield of the cyclodextrin product, a more desirable purity, and a reduction in the amount of by-product waste, as well as by-products that may be useful for other purposes.

[0003] In one aspect, a method for producing a composition comprising cyclodextrin is provided, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with an enzyme capable of converting amylose to cyclodextrin under conditions that allow the conversion of amylose to cyclodextrin, thereby producing a composition comprising cyclodextrin, wherein the enzyme capable of converting amylose to cyclodextrin in (b) is a variant enzyme capable of producing a higher amount and / or concentration of alpha-cyclodextrin than a wild-type enzyme capable of converting amylose to cyclodextrin, and the composition comprising cyclodextrin comprises alpha-cyclodextrin and may further optionally comprise beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof, and the composition comprising cyclodextrin comprises a higher amount and / or concentration of alpha-cyclodextrin than beta-cyclodextrin, gamma-cyclodextrin, or both. Optionally, the enzyme in (a) is amylosucrase or the enzyme mixture in (a) comprises amylosucrase. Optionally, the amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to wild-type amylosucrase. Optionally, the variant amylosucrase is capable of producing a higher amount and / or concentration of amylose from sucrose relative to wild-type amylosucrase. Optionally, the wild-type amylosucrase is Cellulomonas carboniz T26 amylosucrase. Optionally, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 1. Optionally, the wild-type amylosucrase is Neisseria polysaccharea amylosucrase. Optionally, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 2.In some cases, variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In some cases, at least one amino acid variant comprises at least one amino acid substitution relative to wild-type amylosucrase. In some cases, at least one amino acid substitution comprises an amino acid substitution at position 234 relative to wild-type amylosucrase having the amino acid sequence of SEQ ID NO: 2. In some cases, the amino acid substitution at position 234 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H. In some cases, the enzyme mixture of (a) comprises at least two enzymes that can collectively or combinatorially convert sucrose to amylose. In some cases, the enzyme mixture comprises sucrose phosphorylase. In some cases, sucrose phosphorylase can convert sucrose to glucose-1-phosphate. In some cases, contacting (a) further comprises contacting sucrose with sucrose phosphorylase under conditions that allow conversion of sucrose to glucose-1-phosphate. In some cases, sucrose phosphorylase is selected from the group consisting of Bifidobacterium longum sucrose phosphorylase, Leuconostoc mesenteroides sucrose phosphorylase, and Streptococcus mutans sucrose phosphorylase. In some cases, sucrose phosphorylase comprises or consists of an amino acid sequence of any one of SEQ ID NOs: 17-20, or an amino acid sequence having at least about 70% sequence identity with the amino acid sequence of any one of SEQ ID NOs: 17-20. In some cases, the enzyme mixture comprises alpha-glucan phosphorylase. In some cases, alpha-glucan phosphorylase can convert glucose-1-phosphate to amylose.In some cases, contacting in (a) further comprises contacting glucose-1-phosphate with alpha-glucan phosphorylase under conditions that allow conversion from glucose-1-phosphate to amylose. In some cases, the alpha-glucan phosphorylase is selected from the group consisting of Solanum tuberosum alpha-glucan phosphorylase, S. tokodaii strain 7 alpha-glucan phosphorylase, and C. callunae DSM 20145 alpha-glucan phosphorylase. In some cases, the alpha-glucan phosphorylase comprises, or consists of, an amino acid sequence of any one of SEQ ID NOs: 21-24, or an amino acid sequence having at least about 70% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 21-24. In some cases, the enzyme capable of converting amylose to cyclodextrin in (b) comprises a variant cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to the wild-type cyclodextrin glucanotransferase. In some cases, the wild-type cyclodextrin glucanotransferase is Paenibacillus macerans cyclodextrin glucanotransferase. In some cases, the wild-type cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence of any one of SEQ ID NOs: 25-28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 25-28. In some cases, the at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type cyclodextrin glucanotransferase. In some cases, the at least one amino acid substitution comprises an amino acid substitution at amino acid position 146 relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at position 146 is selected from the group consisting of R146A and R146P.In some cases, at least one amino acid substitution includes an amino acid substitution at position 147 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at position 147 is selected from the group consisting of D147P and D147A. In some cases, at least one amino acid substitution includes an amino acid substitution at position 146 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28 and an amino acid substitution at position 147 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at position 146 is selected from the group consisting of R146A and R146P. In some cases, the amino acid substitution at position 147 is selected from the group consisting of D147P and D147A. In some cases, at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28. In some cases, the amino acid substitution at position 372 is D372K. In some cases, at least one amino acid substitution includes an amino acid substitution at position 89 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28. In some cases, the amino acid substitution at position 89 is Y89R. In some cases, at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28 and an amino acid substitution at position 89 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28. In some cases, the amino acid substitution at position 372 is D372K. In some cases, the amino acid substitution at position 89 is Y89R.In some cases, contacting (a), contacting (b), or both, further comprises adding at least one additive that increases the yield of alpha-cyclodextrin relative to beta-cyclodextrin, gamma-cyclodextrin, or both, during (a), (b), or both. In some cases, the at least one additive is CaCl2. In some cases, CaCl2 is added at a concentration of about 1 mM to about 100 mM. In some cases, the at least one additive is ethanol. In some cases, ethanol is added at a concentration of about 1 v / v% to about 10 v / v%. In some cases, contacting (a) and contacting (b) are performed sequentially. In some cases, contacting (a) and contacting (b) are performed simultaneously or substantially simultaneously. In some cases, the amylose produced in (a) is not purified or isolated prior to contacting (b). In some cases, contacting (a), contacting (b), or both are performed in vitro. In some cases, contacting (a), contacting (b), or both are performed in a container, vial, bottle, test tube, well, plate, or encapsulation. In some cases, at least one enzyme of the enzyme or enzyme mixture of (a), the variant enzyme of (b), or both are purified enzymes, isolated enzymes, or both. In some cases, at least one enzyme of the enzyme or enzyme mixture of (a), the variant enzyme of (b), or both are recombinantly produced enzymes. In some cases, contacting (a), contacting (b), or both are performed in vivo. In some cases, contacting (a), contacting (b), or both are performed in a recombinant host cell. In some cases, the recombinant host cell comprises a heterologous nucleic acid encoding at least one enzyme of the enzyme or enzyme mixture of (a), the variant enzyme of (b), or both. In some cases, the recombinant host cell is a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is Escherichia coli.In some cases, at least one enzyme of the enzyme or enzyme mixture of (a), the variant enzyme of (b), or both are produced in Pichia yeast cells such as Pichia pastoris cells. In some cases, the ratio of alpha-cyclodextrin to beta-cyclodextrin in the composition containing cyclodextrin is at least 2:1. In some cases, the ratio of alpha-cyclodextrin to gamma-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

[0004] Incorporation by reference All publications, patents, and patent applications described herein are hereby incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0005] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description, which illustrates exemplary embodiments in which the principles of the invention are utilized, and the accompanying drawings.

Brief Description of the Drawings

[0006]

Figure 1A

Figure 1B

Figure 1C

Figure 2A

Figure 2B

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7A

Figure 7B

[0007] Current cyclodextrin production methods have problems such as supply chain deficiencies, as well as issues related to scalability, quality variability, purification, and the cost of goods. Further, there are several important issues surrounding current cyclodextrin production for use in food and pharmaceuticals, such as, but not limited to, FDA approval of starch after each growth stage and the ability to scale using standard agricultural techniques. The methods of the present disclosure overcome these problems by providing a method for the facile enzymatic synthesis of compositions containing cyclodextrin from sucrose as a starting material, preferably a method for one-pot enzymatic synthesis.

[0008] This specification provides a method for producing a composition comprising cyclodextrin. This specification also provides a method for the enzymatic synthesis of alpha-cyclodextrin. Generally, the methods provided herein do not require the use of starch as a starting material. Preferably, the methods provided herein involve the use of sucrose as a starting material, although in some embodiments, other monosaccharides or disaccharides may be used. This specification also provides a method for the enzymatic conversion of sucrose to alpha-cyclodextrin using various enzymes. The method generally includes the conversion of sucrose to amylose as the first step (step (a)) of the synthetic pathway. In one embodiment, the method involves the use of a single enzyme (e.g., amylosucrase) to convert sucrose to amylose. In another embodiment, the method involves the use of two enzymes (e.g., sucrose phosphorylase and alpha-glucan phosphorylase) to convert sucrose to amylose. The method also generally includes the enzymatic conversion of amylose to alpha-cyclodextrin (e.g., using cyclodextrin glucanotransferase) in the second step (step (b)) of the synthetic pathway. In some embodiments, one or more of the enzymatic steps are performed in vivo (e.g., in a microbial host cell). In some embodiments, one or more of the enzymatic steps are performed in vitro (e.g., in a vessel, vial, bottle, test tube, well, plate, enclosure, using, for example, a purified and / or isolated (e.g., recombinant) enzyme).

[0009] Cyclodextrin is formed by the cyclic arrangement of glucopyranose units conjugated by α1,4 glycosidic bonds. Typically, cyclodextrin is available in three different forms, alpha-cyclodextrin (Figure 1A), beta-cyclodextrin (Figure 1B), and gamma-cyclodextrin (Figure 1C), based on the number of glucose monomers that make up the cyclic arrangement. The number of glucose monomers that make up alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin is 6, 7, and 8, respectively. Cyclodextrin is widely used in the food industry, pharmaceutical industry, and chemical industry because it is low in toxicity, low in immunogenicity, and can form non-covalent complexes with guest molecules. For example, cyclodextrin is widely used as a carrier to improve the water solubility of lipophilic vitamins and hormones. In Western European countries, the intake of natural cyclodextrin is regulated by JECFA (Joint FAO / WHO Expert Committee on Food Additives), and its pharmaceutical applications are under the control of the European Medicines Agency (EMA) in Europe and the Food and Drug Administration (FDA) in the United States. Natural cyclodextrin (CD) can be ingested without being significantly absorbed, so it has received "safe food certification" from the FDA and is generally regarded as a molecule with "GRAS status".

[0010] Alpha-cyclodextrin is widely used in the pharmaceutical industry. Various derivatives of alpha-cyclodextrin have been made to improve the oral bioavailability and solubility of cyclodextrin. For example, modifying the hydroxyl groups of cyclodextrin with alkylhydroxy groups dramatically improves the solubility of cyclodextrin. Some of the possible derivatives include randomly methylated alpha-cyclodextrin and branched alpha-cyclodextrin.

[0011] In one aspect of the present disclosure, a method for producing a composition comprising cyclodextrin is provided. Optionally, the method comprises (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose. Optionally, the method further comprises (b) contacting amylose with an enzyme capable of converting amylose to cyclodextrin under conditions that allow the conversion of amylose to cyclodextrin, thereby producing a composition comprising cyclodextrin. Optionally, the enzyme capable of converting amylose to cyclodextrin is a variant enzyme that can produce alpha-cyclodextrin in an amount and / or concentration (e.g., weight %, mol %, or w / v) higher than that of a wild-type enzyme capable of converting amylose to cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, or both. Optionally, the composition comprising cyclodextrin comprises alpha-cyclodextrin and may further optionally comprise beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof. Optionally, the composition comprising cyclodextrin comprises alpha-cyclodextrin in an amount and / or concentration (e.g., weight %, mol %, or w / v) higher than that of beta-cyclodextrin, gamma-cyclodextrin, or both. Optionally, the amounts and / or concentrations of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin are measured by high performance liquid chromatography (HPLC).

[0012] Method steps (a) for the enzymatic conversion of sucrose to amylose The methods provided herein include the enzymatic conversion of sucrose to amylose. Optionally, the amylose is α-amylose. In some embodiments, the method includes contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that permit the conversion of sucrose to amylose, thereby producing amylose. In one aspect, the method includes the use of a single enzyme for converting sucrose to amylose. In an alternative aspect, the method includes the use of an enzyme mixture (e.g., two enzymes) that collectively or in combination convert sucrose to amylose. Optionally, the sucrose is deuterated sucrose (e.g., one or more hydrogens are replaced with deuterium). Optionally, the sucrose and / or any one or more reagents used in the synthesis reaction are deuterated.

[0013] One-enzyme method for producing amylose from sucrose In some embodiments, the method for converting sucrose to amylose uses a single enzyme. In some cases, the enzyme is amylosucrase. Figure 2A shows a schematic diagram of a single-enzyme method for producing amylose from sucrose. In this example, sucrose is contacted with amylosucrase, which converts the sucrose to amylose. In some cases, the amylosucrase is a wild-type amylosucrase. For example, the wild-type amylosucrase can be the Cellulomonas carboniz T26 amylosucrase (e.g., NCBI accession number N868_11335). In some cases, the wild-type Cellulomonas carboniz T26 amylosucrase can include, or consist of, the amino acid sequence according to SEQ ID NO: 1. In some cases, the wild-type amylosucrase can be the Neisseria polysaccharea amylosucrase (e.g., NCBI accession number AJ011781). In some cases, the wild-type Neisseria polysaccharea amylosucrase can include, or consist of, the amino acid sequence according to SEQ ID NO: 2. Table 1 below shows non-limiting examples of wild-type amylosucrase enzymes (and their amino acid sequences) that can be used in accordance with the methods provided herein.

Table 1

[0014] In some embodiments, the amylosucrase is a variant amylosucrase that includes at least one amino acid variant relative to the wild-type amylosucrase. The variant amylosucrase can include one or more amino acid substitutions, deletions, insertions, and / or modifications relative to the wild-type amylosucrase. In some cases, the variant amylosucrase can produce a higher amount and / or concentration of amylose from sucrose relative to the wild-type amylosucrase.

[0015] In some cases, variant amylosucrase comprises at least one amino acid variant relative to wild-type Cellulomonas carboniz T26 amylosucrase. In some cases, variant amylosucrase comprises at least one amino acid variant relative to SEQ ID NO:1. In some cases, variant amylosucrase comprises at least one amino acid variant relative to wild-type Neisseria polysaccharea amylosucrase. In some cases, variant amylosucrase comprises at least one amino acid variant relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) relative to wild-type Cellulomonas carboniz T26 amylosucrase, preferably at least about 90% sequence identity. In some cases, variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) relative to the amino acid sequence of SEQ ID NO:1, preferably at least about 90% sequence identity.In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to wild-type Neisseria polysaccharea amylosucrase, preferably at least about 90% sequence identity. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity.

[0016] In some cases, at least one amino acid variant contains at least one amino acid substitution relative to the wild-type amylosucrase. In some cases, at least one amino acid variant contains at least one amino acid substitution relative to the wild-type Cellulomonas carboniz T26 amylosucrase. In some cases, at least one amino acid variant contains at least one amino acid substitution relative to the wild-type Neisseria polysaccharea amylosucrase. In some cases, at least one amino acid substitution includes an amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2. In some cases, the amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H. In a preferred embodiment, the amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, and R234K. In this regard, it will be understood that R234Q indicates that the arginine (R) at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is substituted with glutamine (Q), and so on. In some cases, the amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is R234Q (e.g., SEQ ID NO: 3 in Table 2). In some cases, the amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is R234G (e.g., SEQ ID NO: 4 in Table 2). In some cases, the amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is R234A (e.g., SEQ ID NO: 5 in Table 2). In some cases, the amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is R234S (e.g., SEQ ID NO: 6 in Table 2). In some cases, the amino acid substitution at position 234 of the amino acid sequence relative to SEQ ID NO: 2 is R234M (e.g., SEQ ID NO: 7 in Table 2).In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234C (for example, SEQ ID NO: 8 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234K (for example, SEQ ID NO: 9 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234I (for example, SEQ ID NO: 10 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234D (for example, SEQ ID NO: 11 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234Y (for example, SEQ ID NO: 12 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234W (for example, SEQ ID NO: 13 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234E (for example, SEQ ID NO: 14 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234L (for example, SEQ ID NO: 15 in Table 2). In some cases, the amino acid substitution at position 234 for the amino acid sequence of SEQ ID NO: 2 is R234H (for example, SEQ ID NO: 16 in Table 2).

[0017] In some embodiments, the variant amylosucrase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 3 to 16 shown in Table 2. In a preferred embodiment, the variant amylosucrase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 3 to 9 shown in Table 2. In some cases, the variant amylosucrase has at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) compared to an amino acid sequence of any one of SEQ ID NOs: 3 to 16 or 42 shown in Table 2, preferably at least about 90% sequence identity, or comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with an amino acid sequence of any one of SEQ ID NOs: 3 to 16 or 42 shown in Table 2. In a preferred embodiment, the variant amylosucrase has at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) compared to an amino acid sequence of any one of SEQ ID NOs: 3 to 9 shown in Table 2, preferably at least about 90% sequence identity, or comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more).

Table 2-1

Table 2-2

Table 2-3

Table 2-4

Table 2-5

Table 2-6

Table 2-7

[0018] In some embodiments, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 234 relative to SEQ ID NO:2. In this regard, as used throughout this disclosure, the described sequence identity includes amino acid substitutions (i.e., the sequence identity is calculated based on the entire amino acid sequence of the variant enzyme including the amino acid substitutions). Optionally, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 234 relative to SEQ ID NO:2 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H.In a preferred embodiment, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 234 of SEQ ID NO: 2 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, and R234K. Optionally, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234Q of SEQ ID NO: 2. Optionally, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234G of SEQ ID NO: 2.In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234A relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234S relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234M relative to SEQ ID NO:2.In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution R234C relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution R234K relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution R234I relative to SEQ ID NO:2.In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234D relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234Y relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234W relative to SEQ ID NO:2.In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234E relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the aminos. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234H relative to SEQ ID NO:2.

[0019] In some embodiments, the amylosucrase is derived from microbial cells. Optionally, the amylosucrase is isolated and / or purified from the microbial cells. Optionally, the microbial cells are bacterial cells. Optionally, the bacterial cells are Escherichia coli. In some embodiments, the amylosucrase is derived from Neisseria polysaccharea. In some embodiments, the amylosucrase is derived from Cellulomonas carboniz T26. In some embodiments, the amylosucrase can be produced within the microbial cells. In some embodiments, the amylosucrase is expressed within a recombinant host cell (e.g., from a recombinant polynucleotide). Optionally, the amylosucrase is produced recombinantly. Optionally, the amylosucrase is produced in yeast cells (e.g., produced recombinantly). Optionally, the yeast cells are Pichia yeast cells such as Pichia pastoris cells.

[0020] Two-enzyme method for producing amylose from sucrose In some embodiments, the method includes contacting sucrose with an enzyme mixture capable of converting sucrose to amylose under conditions that allow for the conversion of sucrose to amylose, thereby producing amylose. Optionally, the method includes contacting sucrose with an enzyme mixture that includes at least two enzymes that can collectively or combinatorially convert sucrose to amylose. For example, the enzyme mixture can include at least sucrose phosphorylase and alpha-glucan phosphorylase. The method can include contacting sucrose with at least two enzymes simultaneously or substantially simultaneously. Alternatively, the method can include contacting sucrose with at least two enzymes sequentially. Figure 2B shows a schematic of a two-enzyme method for producing amylose from sucrose. In this example, sucrose is contacted with sucrose phosphorylase to convert sucrose to glucose-1-phosphate. Next, glucose-1-phosphate is contacted with alpha-glucan phosphorylase to convert glucose-1-phosphate to amylose. Optionally, sucrose phosphorylase and alpha-glucan phosphorylase are contacted with sucrose simultaneously or substantially simultaneously. In other cases, sucrose phosphorylase and alpha-glucan phosphorylase are added sequentially (e.g., sucrose phosphorylase is first contacted with sucrose to produce glucose-1-phosphate, and then alpha-glucan phosphorylase is added to produce amylose). Optionally, glucose-1-phosphate produced from the reaction with sucrose phosphorylase is isolated and / or purified before contacting it with alpha-glucan phosphorylase. In other cases, glucose-1-phosphate produced from the reaction with sucrose phosphorylase is not isolated and / or purified before contacting it with alpha-glucan phosphorylase. As used in the context of adding two or more components to the reaction mixture described herein, the term "substantially simultaneously" means that the two or more components are added to the reaction mixture within 10 seconds of each other.

[0021] In some cases, the sucrose phosphorylase is a wild-type sucrose phosphorylase. For example, the wild-type sucrose phosphorylase can be Bifidobacterium longum sucrose phosphorylase (e.g., NCBI accession number AAO84039). In some cases, the wild-type Bifidobacterium longum sucrose phosphorylase can have the amino acid sequence according to SEQ ID NO: 17. In some cases, the wild-type sucrose phosphorylase can be Leuconostoc mesenteroide sucrose phosphorylase (e.g., NCBI accession number D90314.1). In some cases, the wild-type Leuconostoc mesenteroide sucrose phosphorylase can have the amino acid sequence according to SEQ ID NO: 18. In some cases, the wild-type sucrose phosphorylase can be Streptococcus mutans sucrose phosphorylase (e.g., NCBI accession number NZ_CP013237.1). In some cases, the wild-type Streptococcus mutans sucrose phosphorylase can have the amino acid sequence according to SEQ ID NO: 19 (e.g., NCBI accession number P10249). In some cases, the sucrose phosphorylase enzyme is a variant sucrose phosphorylase enzyme. In some cases, the variant sucrose phosphorylase has one or more amino acid substitutions relative to the wild-type sucrose phosphorylase. In some cases, the variant sucrose phosphorylase has amino acid substitutions in one or all of the amino acid residues T47, S62, Y77, V128, K140, Q144, N155, and D249 relative to SEQ ID NO: 19. In some cases, the amino acid substitution at amino acid position 47 relative to SEQ ID NO: 19 is T47S. In some cases, the amino acid substitution at amino acid position 62 relative to SEQ ID NO: 19 is S62P. In some cases, the amino acid substitution at amino acid position 77 relative to SEQ ID NO: 19 is Y77H. In some cases, the amino acid substitution at amino acid position 128 relative to SEQ ID NO: 19 is V128L. In some cases, the amino acid substitution at amino acid position 140 relative to SEQ ID NO: 19 is K140M.In some cases, the amino acid substitution at position 144 for SEQ ID NO: 19 is Q144R. In some cases, the amino acid substitution at position 155 for SEQ ID NO: 19 is N155S. In some cases, the amino acid substitution at position 249 for SEQ ID NO: 19 is D249G. In some cases, the variant sucrose phosphorylase has amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G with respect to SEQ ID NO: 19. In some cases, the variant sucrose phosphorylase enzyme comprises or consists of the amino acid sequence according to SEQ ID NO: 20. Table 3 below shows non-limiting examples of sucrose phosphorylase enzymes (and their amino acid sequences) that can be used according to the methods provided herein.

Table 3-1

Table 3-2

[0022] In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the wild-type Bifidobacterium longum sucrose phosphorylase, preferably at least about 90% sequence identity. In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 17, preferably at least about 90% sequence identity. In some cases, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the wild-type Leuconostoc mesenteroides sucrose phosphorylase, preferably at least about 90% sequence identity.In some cases, sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 18, preferably at least about 90% sequence identity. In some cases, sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with wild-type Streptococcus mutans sucrose phosphorylase, preferably at least about 90% sequence identity. In some cases, sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 19, preferably at least about 90% sequence identity.In some cases, sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 20, preferably at least about 90% sequence identity, and comprises the amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G relative to SEQ ID NO: 19.

[0023] In some embodiments, sucrose phosphorylase is derived from a microbial cell. In some cases, sucrose phosphorylase is isolated and / or purified from a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, the bacterial cell is Escherichia coli. In some embodiments, sucrose phosphorylase is derived from Bifidobacterium longum. In some embodiments, sucrose phosphorylase is derived from Leuconostoc mesenteroides. In some embodiments, sucrose phosphorylase is derived from Streptococcus mutans. In some embodiments, sucrose phosphorylase can be produced within a microbial cell. In some embodiments, sucrose phosphorylase is expressed in a recombinant host cell (e.g., from a recombinant polynucleotide). In some cases, sucrose phosphorylase is produced recombinantly. In some cases, sucrose phosphorylase is produced in a yeast cell (e.g., produced recombinantly). In some cases, the yeast cell is a Pichia yeast cell such as a Pichia pastoris cell.

[0024] In some embodiments, the alpha - glucan phosphorylase is a wild - type alpha - glucan phosphorylase. In some cases, the wild - type alpha - glucan phosphorylase can be a Solanum tuberosum alpha - glucan phosphorylase (e.g., NCBI accession number D00520.1). In some cases, the wild - type Solanum tuberosum alpha - glucan phosphorylase can have the amino acid sequence according to SEQ ID NO: 21. In some cases, the wild - type alpha - glucan phosphorylase can be an S. tokodaii strain 7 alpha - glucan phosphorylase (e.g., NCBI accession number NC_003106.2). In some cases, the wild - type S. tokodaii strain 7 alpha - glucan phosphorylase can have the amino acid sequence according to SEQ ID NO: 22. In some cases, the wild - type alpha - glucan phosphorylase can be a C. callunae DSM 20145 alpha - glucan phosphorylase (e.g., NCBI accession number AY102616.1). In some cases, the wild - type C. callunae DSM 20145 alpha - glucan phosphorylase can have the amino acid sequence according to SEQ ID NO: 23. In some cases, the alpha - glucan phosphorylase enzyme is a variant alpha - glucan phosphorylase enzyme. In some cases, the variant alpha - glucan phosphorylase has one or more amino acid substitutions relative to the wild - type alpha - glucan phosphorylase. In some cases, the variant alpha - glucan phosphorylase has amino acid substitutions in one or all of the amino acid residues F39, N135, and T706 relative to SEQ ID NO: 21. In some cases, the amino acid substitution at position 39 of the amino acid relative to SEQ ID NO: 21 is F39L. In some cases, the amino acid substitution at position 135 of the amino acid relative to SEQ ID NO: 21 is N135S. In some cases, the amino acid substitution at position 706 of the amino acid relative to SEQ ID NO: 21 is T706I. In some cases, the variant alpha - glucan phosphorylase has the amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO: 21.In some cases, the variant alpha - glucan phosphorylase enzyme has the amino acid sequence according to SEQ ID NO: 24. Table 4 below shows non - limiting examples of alpha - glucan phosphorylase enzymes (and their amino acid sequences) that can be used according to the methods provided herein.

Table 4 - 1

Table 4 - 2

[0025] In some cases, the alpha - glucan phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the wild - type Solanum tuberosum alpha - glucan phosphorylase, preferably at least about 90% sequence identity. In some cases, the alpha - glucan phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 21, preferably at least about 90% sequence identity. In some cases, the alpha - glucan phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the wild - type S. tokodaii strain 7 alpha - glucan phosphorylase, preferably at least about 90% sequence identity.In some cases, the alpha - glucan phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 22, preferably at least about 90% sequence identity. In some cases, the alpha - glucan phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the wild - type C. callunae DSM 20145 alpha - glucan phosphorylase, preferably at least about 90% sequence identity. In some cases, the alpha - glucan phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 23, preferably at least about 90% sequence identity.In some cases, sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 21, preferably at least about 90% sequence identity, and includes the amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO: 21.

[0026] In some embodiments, the alpha - glucan phosphorylase is derived from microbial cells. In some cases, the alpha - glucan phosphorylase is isolated and / or purified from microbial cells. In some cases, the microbial cells are bacterial cells. In some cases, the bacterial cells are Escherichia coli. In some embodiments, the alpha - glucan phosphorylase is derived from Solanum tuberosum. In some embodiments, the alpha - glucan phosphorylase is derived from the S. tokodaii strain 7. In some embodiments, the alpha - glucan phosphorylase is derived from C. callunae DSM 20145. In some embodiments, the alpha - glucan phosphorylase can be produced within microbial cells. In some embodiments, the alpha - glucan phosphorylase is expressed in a recombinant host cell (e.g., from a recombinant polynucleotide). In some cases, the alpha - glucan phosphorylase is recombinantly produced. In some cases, the alpha - glucan phosphorylase is produced in yeast cells (e.g., recombinantly produced). In some cases, the yeast cells are Pichia yeast cells such as Pichia pastoris cells.

[0027] Method steps (b) for the enzymatic conversion of amylose to alpha-cyclodextrin In various embodiments, the method further comprises enzymatically converting amylose (e.g., produced by the methods provided herein (e.g., method step (a))) to cyclodextrin, preferably alpha-cyclodextrin. Optionally, the method comprises contacting amylose with an enzyme or enzyme mixture (e.g., two or more enzymes, etc.) capable of converting amylose to cyclodextrin under conditions that permit the conversion of amylose to cyclodextrin. Optionally, the enzyme capable of converting amylose to cyclodextrin is a variant enzyme that can produce alpha-cyclodextrin in a higher amount and / or concentration than beta-cyclodextrin, gamma-cyclodextrin, or both, relative to a wild-type enzyme capable of converting amylose to cyclodextrin.

[0028] In some embodiments, the enzyme capable of converting amylose to cyclodextrin comprises a variant cyclodextrin glucanotransferase. Optionally, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type cyclodextrin glucanotransferase. Figure 3 shows the enzymatic conversion of amylose to alpha-cyclodextrin using cyclodextrin glucanotransferase. Preferably, the cyclodextrin glucanotransferase produces alpha-cyclodextrin from amylose in a higher amount and / or concentration than the amount and / or concentration of beta-cyclodextrin and / or gamma-cyclodextrin.

[0029] In some embodiments, the cyclodextrin glucanotransferase is a variant cyclodextrin glucanotransferase comprising at least one amino acid variant relative to the wild-type cyclodextrin glucanotransferase. The variant cyclodextrin glucanotransferase can include one or more amino acid substitutions, deletions, insertions, and / or modifications relative to the wild-type cyclodextrin glucanotransferase. Optionally, the variant cyclodextrin glucanotransferase can produce, from amylose, a higher amount and / or concentration of alpha-cyclodextrin compared to beta-cyclodextrin and / or gamma-cyclodextrin relative to the wild-type cyclodextrin glucanotransferase.

[0030] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type Paenibacillus macerans cyclodextrin glucanotransferase (e.g., NCBI accession number AAA22298.1 or X59045.1; e.g., SEQ ID NOs: 25-28). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to any one of SEQ ID NOs: 25-28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of wild-type Paenibacillus macerans cyclodextrin glucanotransferase, preferably at least about 90% sequence identity. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of any one of SEQ ID NOs: 25-28, preferably at least about 90% sequence identity.

[0031] In some cases, at least one amino acid variant contains at least one amino acid substitution relative to wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution includes an amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146A (e.g., SEQ ID NO: 29 in Table 5). In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146P (e.g., SEQ ID NO: 30 in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147A (e.g., SEQ ID NO: 31 in Table 5). In some cases, the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147P (e.g., SEQ ID NO: 32 in Table 5). In some cases, at least one amino acid substitution includes amino acid substitutions at amino acid positions 146 and 147 relative to the amino acid sequence of SEQ ID NO: 28. In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146A and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147P (e.g., SEQ ID NO: 33 in Table 5). In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146P and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147A (e.g., SEQ ID NO: 34 in Table 5). In some cases, the amino acid substitution at amino acid position 146 relative to the amino acid sequence of SEQ ID NO: 28 is R146P and the amino acid substitution at amino acid position 147 relative to the amino acid sequence of SEQ ID NO: 28 is D147P (e.g., SEQ ID NO: 35 in Table 5).

[0032] In some cases, at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28. In some cases, the amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28 is D372K (e.g., SEQ ID NO: 36 (for SEQ ID NO: 26) and SEQ ID NO: 39 (for SEQ ID NO: 28) in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28. In some cases, the amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28 is Y89R (e.g., SEQ ID NO: 37 (for SEQ ID NO: 26) and SEQ ID NO: 40 (for SEQ ID NO: 28) in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28 and an amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 26 or SEQ ID NO: 28. In some cases, the amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 26 or 28 is D372K, and the amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 26 or 28 is Y89R (e.g., SEQ ID NO: 38 (for SEQ ID NO: 26) and SEQ ID NO: 41 (for SEQ ID NO: 28) in Table 5).

[0033] In some embodiments, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 25 to 41 shown in Table 5. In some embodiments, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) sequence identity, preferably at least about 90% sequence identity, with an amino acid sequence according to any one of SEQ ID NOs: 25 to 41 shown in Table 5.

[0034] In certain embodiments, the cyclodextrin glucanotransferase comprises or consists of, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence according to SEQ ID NO: 28.

[0035] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 33, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) sequence identity with the amino acid sequence according to SEQ ID NO: 33, preferably at least about 90% sequence identity.

[0036] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 34, or comprises or consists of an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) sequence identity with the amino acid sequence according to SEQ ID NO: 34, preferably at least about 90% sequence identity.

[0037] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 35, or has an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) sequence identity with the amino acid sequence according to SEQ ID NO: 35, preferably at least about 90% sequence identity, and comprises or consists of such an amino acid sequence.

[0038] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 41, or has an amino acid sequence having at least about 70% (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) sequence identity with the amino acid sequence according to SEQ ID NO: 41, preferably at least about 90% sequence identity, and comprises or consists of such an amino acid sequence.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

Table 5-6

Table 5-7

Table 5-8

[0039] In some embodiments, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and an amino acid substitution R146A relative to SEQ ID NO: 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and an amino acid substitution R146P relative to SEQ ID NO: 28.

[0040] In some embodiments, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and an amino acid substitution D147P relative to SEQ ID NO: 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, and an amino acid substitution D147A relative to SEQ ID NO: 28.

[0041] In some embodiments, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 28, and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, the amino acid substitution R146A relative to SEQ ID NO: 28, and the amino acid substitution D147P relative to SEQ ID NO: 28.In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 28, and an amino acid substitution D147A relative to SEQ ID NO: 28. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 28, preferably at least about 90% sequence identity, an amino acid substitution R146P relative to SEQ ID NO: 28, and an amino acid substitution D147P relative to SEQ ID NO: 28.

[0042] In some embodiments, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 26 or 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, and an amino acid sequence having the amino acid substitution D372K relative to SEQ ID NO: 26 or 28.

[0043] In some embodiments, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 26 or 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, and an amino acid sequence having the amino acid substitution Y89R relative to SEQ ID NO: 26 or 28.

[0044] In some embodiments, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 26 or 28, and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 26 or 28. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26 or 28, preferably at least about 90% sequence identity, the amino acid substitution D372K relative to SEQ ID NO: 26 or 28, and the amino acid substitution Y89R relative to SEQ ID NO: 26 or 28.

[0045] In some embodiments, cyclodextrin glucanotransferase is derived from microbial cells. In some cases, cyclodextrin glucanotransferase is isolated and / or purified from microbial cells. In some cases, the microbial cells are bacterial cells. In some cases, the bacterial cells are Escherichia coli. In some embodiments, cyclodextrin glucanotransferase is derived from Paenibacillus macerans. In some embodiments, cyclodextrin glucanotransferase can be produced within microbial cells. In some embodiments, cyclodextrin glucanotransferase is expressed within recombinant host cells (e.g., from a recombinant polynucleotide). In some cases, cyclodextrin glucanotransferase is produced recombinantly. In some cases, cyclodextrin glucanotransferase is produced in yeast cells (e.g., produced recombinantly). In some cases, the yeast cells are Pichia yeast cells such as Pichia pastoris cells.

[0046] In various embodiments, one or more additives can be added to the reaction mixture. It will be understood that the reaction mixture means the reaction mixture present in method step (a), or method step (b), or both method steps (a) and (b). When more than one additive is used, the additives can be introduced simultaneously or sequentially. In some cases, one or more additives can increase the amount and / or concentration of alpha-cyclodextrin produced compared to the same reaction without using one or more additives. In some cases, one or more additives can increase the ratio of alpha-cyclodextrin to beta-cyclodextrin, gamma-cyclodextrin, or both compared to the same reaction without using one or more additives. In some cases, one or more additives include calcium chloride (CaCl2). In some cases, one or more additives include ethanol. In some cases, one or more additives include both CaCl2 and ethanol. CaCl2 can be added at a concentration of about 1 mM to about 100 mM. In some cases, CaCl2 is added at a concentration of about 10 mM. Ethanol can be added at a concentration of about 1 v / v% to about 10 v / v%. In some cases, ethanol is added at a concentration of about 2 v / v%.

[0047] In various embodiments, the methods provided herein produce alpha-cyclodextrin at a higher ratio than beta-cyclodextrin, gamma-cyclodextrin, or both. For example, in some cases, the methods provided herein provide a ratio of alpha-cyclodextrin to beta-cyclodextrin, gamma-cyclodextrin, or both of at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, or more. In certain embodiments, the methods provided herein provide a ratio of alpha-cyclodextrin to beta-cyclodextrin of at least 1.25:1. For example, the ratio can be at least 1.5:1, at least 1.75:1, at least 2:1, at least 2.25:1, at least 2.5:1, at least 2.75:1, at least 3:1, or more. In certain embodiments, the methods provided herein provide a ratio of alpha-cyclodextrin to gamma-cyclodextrin of at least 2:1. For example, the ratio can be at least 3:1, at least 5:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 75:1, at least 100:1, or more. In certain embodiments, the methods provided herein provide a ratio of alpha-cyclodextrin to both beta-cyclodextrin and gamma-cyclodextrin of at least 1.25:1. For example, the ratio can be at least 1.3:1, at least 1.4:1, at least 1.5:1, at least 1.6:1, at least 1.7:1, at least 1.8:1, at least 1.9:1, at least 2:1, at least 3:1, or more.

[0048] Throughout the present disclosure, methods are outlined for achieving robust enzyme activity at each step to obtain alpha-cyclodextrin in a higher yield than currently achievable yields. In some embodiments, a first enzymatic step of converting sucrose to amylose (e.g., as described herein) is carried out over a first period, thereby enabling a catalytic conversion of sucrose to amylose, followed by a second enzymatic step of converting amylose to alpha-cyclodextrin (e.g., as described herein) over a second period, thereby enabling a catalytic conversion of amylose to alpha-cyclodextrin. In some embodiments, the first enzymatic reaction (e.g., as described herein, e.g., converting sucrose to amylose) and the second enzymatic reaction (e.g., as described herein, e.g., converting amylose to alpha-cyclodextrin) are carried out in the same reservoir (e.g., one-pot synthesis method).

[0049] In some embodiments, the first period is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 85 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, at least 180 minutes, at least 195 minutes, at least 210 minutes, at least 225 minutes, at least 240 minutes, at least 255 minutes, at least 270 minutes, at least 285 minutes, or at least 300 minutes. In some embodiments, the second period is at least 30 minutes, at least 45 minutes, at least 60 minutes, at least 85 minutes, at least 90 minutes, at least 105 minutes, at least 120 minutes, at least 135 minutes, at least 150 minutes, at least 165 minutes, at least 180 minutes, at least 195 minutes, at least 210 minutes, at least 225 minutes, at least 240 minutes, at least 255 minutes, at least 270 minutes, at least 285 minutes, or at least 300 minutes. In some embodiments, the first period is shorter than the second period. In some embodiments, the first period is longer than the second period. In some embodiments, the first period is the same or substantially the same length as the second period. In some embodiments, sucrose is added to the reaction reservoir batch by batch. In some embodiments, the enzyme used in the first enzyme reaction step (e.g., as described herein, e.g., to convert sucrose to amylose) is added once at the start of the reaction period and then readded after a certain period of time has elapsed to promote catalytic activity. In some embodiments, sucrose is added once at the start of the reaction period and then readded after a certain period of time has elapsed to replenish the sucrose. In some embodiments, the enzyme used in the first enzyme reaction step (e.g., as described herein, e.g., to convert sucrose to amylose) is added to the same reaction reservoir at the same time as the enzyme used in the second enzyme reaction step (e.g., to convert amylose to alpha-cyclodextrin).In some embodiments, the enzyme used in the first enzyme reaction step (e.g., as described herein, e.g., to convert sucrose to amylose) is added at a different time (e.g., prior thereto) than the enzyme used in the second enzyme reaction step (e.g., to convert amylose to alpha-cyclodextrin).

[0050] In some embodiments, the sucrose concentration is maximized for efficient conversion to amylose. In some embodiments, the starting concentration of sucrose in the reaction is at least about 50 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 100 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 150 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 200 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 250 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 300 g / L. In some embodiments, the starting concentration of sucrose in the reaction is at least about 350 g / L.

[0051] In some embodiments, the reaction time is an important consideration for obtaining the maximum yield of alpha-cyclodextrin. In some embodiments, the production of alpha-cyclodextrin can involve the decomposition of the product into glucose, maltose, and other sugars. Therefore, it is important to obtain alpha-cyclodextrin without decomposing it. In some embodiments, the entire reaction (e.g., method step (a) and method step (b)) is carried out over 12 hours or less. In some embodiments, the entire reaction (e.g., method step (a) and method step (b)) is carried out over 8 hours or less. In some embodiments, the entire reaction is carried out over 7 hours or less. In some embodiments, the entire reaction is carried out over 6 hours or less. In some embodiments, the entire reaction is carried out over 5 hours or less. In some embodiments, the entire reaction is carried out over 4 hours or less. In some embodiments, the entire reaction is carried out over 3 hours or less. In some embodiments, the entire reaction is carried out over 2 hours or less. In some embodiments, the entire reaction is carried out over 1 hour or less.

[0052] Temperature is an important consideration for maximizing the yield of alpha-cyclodextrin. In some embodiments, one or more of the enzymatic reactions are carried out at about 30°C to about 55°C, such as about 40°C to about 50°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 40°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 41°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 42°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 43°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 44°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 45°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 46°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 47°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 48°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 49°C. In some embodiments, one or more of the enzymatic reactions are carried out at about 50°C. Preferably, one or more of the reactions are carried out at about 45°C.

[0053] In some embodiments, the enzymatic reaction of step (a) is carried out at about 40°C to about 55°C, such as about 45°C to about 50°C. In some embodiments, the enzymatic reaction of step (b) is carried out at about 40°C to about 50°C. Step (a) and step (b) may be carried out at different temperatures, or preferably, step (a) and step (b) are carried out at substantially the same temperature. If step (a) involves the use of a single enzyme (e.g., amylosucrase), the enzymatic reaction of step (a) is preferably carried out at about 45°C. In this embodiment, the enzymatic reaction of step (b) is also preferably carried out at about 45°C. If step (a) involves the use of at least two enzymes (e.g., sucrose phosphorylase and alpha-glucan phosphorylase), the enzymatic reaction of step (a) is preferably carried out at about 45°C or about 50°C. In this embodiment, the enzymatic reaction of step (b) is also preferably carried out at about 45°C or about 50°C, respectively.

[0054] In one-pot synthesis, even though the optimal temperature of each enzyme may vary slightly, it is considered that the functionality of the enzyme mixture(s) should be maximized.

[0055] In some embodiments, the reaction is carried out at a pH of 5.0 to 9.0, for example, 6.0 to 8.0, for example, 6.5 to 7.5. In some embodiments, the reaction is carried out at a pH of 6.0. In some embodiments, the reaction is carried out at a pH of 7.0. In some embodiments, the reaction is carried out at a pH of 8.0.

[0056] In some embodiments, one or more of the enzyme reactions are carried out in a reaction mixture containing a buffer. Any suitable buffer known in the art may be used. For example, the buffer may be selected from the group consisting of sodium citrate, disodium hydrogen phosphate, and Tris-HCl. The buffer may be present in the mixture at a concentration of about 50 mM to about 200 mM. In a preferred embodiment, the buffer is present at a concentration of about 100 mM.

[0057] In some embodiments, the reaction is carried out in a reservoir having a reservoir volume of about 1 mL to about 1,000,000 L. For example, the reaction may be carried out in a reservoir having a reservoir volume of about 100 mL to about 10 L, such as a reservoir volume of about 500 mL or about 10 L.

[0058] In some embodiments, the total reaction volume is from about 1 mL to about 1,000,000 L. For example, the total reaction volume can be from about 100 mL to about 10 L, such as a total reaction volume of about 500 mL or about 5 L. In some embodiments, the total reaction volume is less than the reservoir volume. For example, in a reaction carried out in a reservoir having a reservoir volume of about 10 L, a total reaction volume of about 5 L can be used.

[0059] In some embodiments, the reaction is carried out in a stirred tank reactor (STR), a loop reactor, a plug flow reactor, a single-stage or multi-stage continuous stirred tank reactor, or any other suitable reactor known in the art. In some embodiments, the reaction is carried out in a stirred tank reactor and the reactants are stirred at about 100 to about 200 rpm, such as about 160 rpm.

[0060] In some embodiments, one or more of the enzymatic reactions are carried out in a reaction mixture containing an organic solvent, preferably toluene. The reaction mixture preferably also contains water. Without wishing to be bound by any theory set forth herein, the inventors have confirmed that the addition of an organic solvent surprisingly increases the yield of alpha-cyclodextrin obtained from the enzymatic reaction. For example, the addition of an organic solvent can increase the yield of alpha-cyclodextrin by at least about 5%, such as at least about 10%, such as at least about 15%, such as at least about 20%, such as at least about 50%, such as at least about 100%, such as at least about 150%, such as at least about 200%, such as at least about 250%, such as at least about 300%, such as at least about 350%, such as at least about 400% compared to the yield obtained from the enzymatic reaction carried out without an organic solvent. It is believed that the addition of the organic solvent reduces the solubility of alpha-cyclodextrin in the reaction mixture, causing alpha-cyclodextrin to precipitate and reducing the concentration of alpha-cyclodextrin in the reaction mixture, thereby increasing the yield of alpha-cyclodextrin. This prevents the degradation of alpha-cyclodextrin by the enzyme.

[0061] In some embodiments, the amount of organic solvent (preferably toluene) in the reaction mixture is about 0.1 v / v% to about 40 v / v% of the reaction mixture, such as about 1 v / v% to about 35 v / v%, such as about 5 v / v% to about 25 v / v%.

[0062] In some embodiments, the organic solvent is introduced at the start or during the enzymatic reaction of step (a). In some preferred embodiments, the organic solvent is introduced at the start or during the enzymatic reaction of step (b). For example, in embodiments where the entire reaction (e.g., method step (a) and method step (b)) is carried out over 8 hours or less, the organic solvent can be introduced about 1 hour after the start of the enzymatic reaction (b).

[0063] In some embodiments, the enzyme used in step (a) is amylosucrase. In some embodiments, the starting concentration of amylosucrase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL.

[0064] In some embodiments, the enzyme mixture used in step (a) contains sucrose phosphorylase and alpha - glucan phosphorylase. In some embodiments, the starting concentration of sucrose phosphorylase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL. In some embodiments, the starting concentration of alpha - glucan phosphorylase in the reaction mixture is about 1 to about 30 U / mL, such as about 5 to about 25 U / mL, such as about 8 to about 25 U / mL.

[0065] In some embodiments, the enzyme is provided in the whole cell lysate. Preferably, the ratio of the starting concentration of the enzyme in step (b) (measured as the volume in the whole cell lysate) to the enzyme in step (a) is about 1:1 to about 50:1, such as about 2:1 to about 50:1, such as about 5:1 to about 40:1, such as about 10:1 to about 30:1. In a preferred embodiment, the ratio is about 20:1.

[0066] In certain embodiments, any one of the enzyme reactions provided herein (e.g., the first enzyme reaction that converts sucrose to amylose and / or the second enzyme reaction that converts amylose to alpha-cyclodextrin) can occur within a microbial host cell. Optionally, the microbial cell is a bacterial cell. Optionally, the bacterial cell is Escherichia coli. For example, the microbial host cell can contain one or more heterologous nucleic acid molecules encoding one or more of the enzymes provided herein. The microbial host cell can express one or more of the enzymes provided herein. Optionally, sucrose and / or one or more intermediates of the enzyme reaction can be fed to the microbial host cell. For example, sucrose can be fed to the microbial host cell, and the conversion from sucrose to alpha-cyclodextrin can occur within the microbial host cell.

[0067] In some embodiments, one or more of the enzymes used in the enzyme reactions provided herein may be immobilized on a resin. For example, the enzyme may be covalently bound to the resin. Alternatively, the enzyme may be non-covalently bound to the resin. For example, the enzyme may be linked to Ni resin via a His tag. For example, the enzyme of (a) can be a variant amylosucrase (e.g., this variant amylosucrase can contain or consist of the amino acid sequence according to SEQ ID NO: 3), and the enzyme may be immobilized on a resin. Alternatively, or additionally, the enzyme of (b) can be a variant cyclodextrin glucanotransferase, and the enzyme may be immobilized on a resin. Optionally, the enzyme or enzyme mixture of (a) and the enzyme of (b) are immobilized on the same resin.

[0068] The resin-immobilized enzyme can be reused by the method described in this specification. However, the inventors have found that when the resin-immobilized enzyme is reused, the yield of alpha-cyclodextrin tends to decrease. This is presumably due to the enzyme leaching from the resin during use, resulting in a decrease in the enzyme conversion rate. Therefore, it is desirable to improve the enzyme stability on the resin and thereby prevent enzyme leaching. This is because it enables the resin-immobilized enzyme to be reused more frequently and / or at a higher enzyme conversion rate, thereby increasing the yield of the reaction.

[0069] The inventors have found that enzyme stability can be improved by using freeze-dried enzymes, spray-drying the enzymes, and / or introducing stabilizing compounds.

[0070] In some embodiments, the enzyme is provided in a cell slurry or in a whole cell lysate. For example, a cell slurry containing recombinant cells expressing the enzyme can be suspended, lysed, and centrifuged in a buffer (such as sodium citrate buffer) to provide a whole cell lysate containing the enzyme. Methods of cell lysis are known in the art. By way of example, cells can be lysed by homogenization, chemical lysis, sonication, freeze / thaw, lytic enzymes, acid lysis, and / or alkaline lysis. In a preferred embodiment, the cells are lysed by homogenization.

[0071] In some embodiments, the cell slurry or whole cell lysate further comprises a stabilizing compound. In some embodiments, the stabilizing compound is selected from the group consisting of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, and combinations thereof. In some embodiments, the stabilizing compound is added in an amount of about 0.1 w / v% to about 10 w / v%, such as about 0.5 w / v% to about 5 w / v% of the cell slurry or whole cell lysate. For example, the stabilizing compound is added at 0.5 w / v%, 1.0 w / v%, or 5 w / v% of the cell slurry or whole cell lysate. In preferred embodiments, the stabilizing compound is mannitol, sorbitol, sucrose, or combinations thereof.

[0072] In some embodiments, the cell slurry or cell lysate may be lyophilized. For example, the cell slurry or cell lysate may be lyophilized over a period of two days. Methods of lyophilization are known in the art.

[0073] The inventors have found that adding a stabilizing compound to the cell slurry or whole cell lysate (as described above) increases the enzyme stability compared to the cell slurry or whole cell lysate without the stabilizing compound, and that lyophilizing the cell slurry or whole cell lysate (as described above) increases the enzyme stability compared to the non-lyophilized cell slurry or whole cell lysate. The cell slurry or cell lysate may be resuspended and shaken to redissolve before use in the methods described herein.

[0074] In some embodiments, the methods described herein produce a composition comprising at least 2 g / L of alpha-cyclodextrin. In some embodiments, the method produces a composition comprising at least 3 g / L of alpha-cyclodextrin, at least 4 g / L of alpha-cyclodextrin, at least 5 g / L of alpha-cyclodextrin, at least 6 g / L of alpha-cyclodextrin, at least 7 g / L of alpha-cyclodextrin, at least 8 g / L of alpha-cyclodextrin, at least 9 g / L of alpha-cyclodextrin, at least 10 g / L of alpha-cyclodextrin, at least 12 g / L of alpha-cyclodextrin, at least 15 g / L of alpha-cyclodextrin, at least 20 g / L of alpha-cyclodextrin, at least 30 g / L of alpha-cyclodextrin, at least 40 g / L of alpha-cyclodextrin, at least 50 g / L of alpha-cyclodextrin, or at least 60 g / L of alpha-cyclodextrin. In a preferred embodiment, the method produces a composition comprising at least 10 g / L of alpha-cyclodextrin.

[0075] In some embodiments, the percent yield of alpha-cyclodextrin is at least about 10%, such as at least about 20%, such as at least about 30%, such as at least about 40%, such as at least about 50%, or such as at least about 60%, and this percent yield is calculated by dividing the total amount of alpha-cyclodextrin produced by the methods described herein by the maximum theoretical amount of alpha-cyclodextrin that could be produced from the starting sucrose reagent.

[0076] Also, in the present specification, a composition containing cyclodextrin is provided, the cyclodextrin includes alpha-cyclodextrin, and may further include beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof as appropriate. The composition containing cyclodextrin includes alpha-cyclodextrin in an amount and / or concentration higher than that of beta-cyclodextrin, gamma-cyclodextrin, or both. Preferably, the composition is obtained from the method provided in the present specification. Preferably, the composition does not contain beta-cyclodextrin and / or gamma-cyclodextrin. Preferably, the ratio of alpha-cyclodextrin in the composition to beta-cyclodextrin, gamma-cyclodextrin, or both is at least 2:1, at least 3:1, at least 4:1, at least 5:1, at least 6:1, at least 7:1, at least 8:1, at least 9:1, at least 10:1, at least 20:1, at least 30:1, at least 40:1, at least 50:1, at least 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, or more.

[0077] In a preferred embodiment, the present invention provides a method for producing a composition comprising cyclodextrin, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with cyclodextrin glucanotransferase, thereby producing a composition comprising cyclodextrin, wherein the cyclodextrin glucanotransferase in (b) is a variant enzyme capable of producing alpha-cyclodextrin in an amount and / or concentration higher than that of beta-cyclodextrin, gamma-cyclodextrin, or both, relative to a wild-type enzyme capable of converting amylose to cyclodextrin, the composition comprising cyclodextrin comprises alpha-cyclodextrin and may further optionally comprise beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof, the ratio of alpha-cyclodextrin in the composition to beta-cyclodextrin, gamma-cyclodextrin, or both is at least 2:1, steps (a) and (b) are carried out simultaneously, steps (a) and (b) are carried out at about 45°C to about 55°C, steps (a) and (b) are carried out at a pH of about 7.0 to about 7.5, steps (a) and (b) are carried out in a reaction mixture comprising water, ethanol, CaCl2, and an optional organic solvent (preferably toluene), and the entire reaction is carried out over a period of 8 hours or less.

[0078] Also provided herein is alpha-cyclodextrin. Preferably, the alpha-cyclodextrin is obtained from the method provided herein.

[0079] Also provided herein is the use of sucrose as a starting material for the production of alpha-cyclodextrin. Also provided herein is the use of sucrose in a method for producing alpha-cyclodextrin, the method not using starch.

[0080] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for converting sucrose to amylose, which can convert sucrose to amylose as described herein.

[0081] Also provided herein is the use of any one of the variant enzymes described herein for converting amylose to cyclodextrin and / or for producing alpha-cyclodextrin in an amount and / or concentration higher than that of beta-cyclodextrin, gamma-cyclodextrin, or both, which can convert amylose to cyclodextrin as described herein.

[0082] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for the production of alpha-cyclodextrin, which does not require starch as a starting material.

[0083] Also provided herein is any one of the enzymes or enzyme mixtures described herein. For example, provided herein are enzymes comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 1-42. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with any one of the amino acid sequences of SEQ ID NOs: 1-42.

[0084] Preferably, the enzyme is a variant amylosucrase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 3-16 or 42. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with any one of the amino acid sequences of SEQ ID NOs: 3-16 or 42.

[0085] Preferably, this enzyme is a variant sucrose phosphorylase enzyme comprising or consisting of the amino acid sequence of SEQ ID NO: 20. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence of SEQ ID NO: 20.

[0086] Preferably, this enzyme is a variant alpha-glucan phosphorylase enzyme comprising or consisting of the amino acid sequence of SEQ ID NO: 24. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence of SEQ ID NO: 24.

[0087] Preferably, this enzyme is a variant cyclodextrin glucanotransferase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 29 to 41. Also provided herein are enzymes comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with any one of the amino acid sequences of SEQ ID NOs: 29 to 41.

[0088] Also provided herein are enzyme compositions comprising one or more of the enzymes described herein.

[0089] Also provided herein are genes encoding any one of the variant enzymes described herein. Also provided herein are vectors encoding any one of the variant enzymes described herein. Also provided herein are recombinant host cells comprising any one of the genes, vectors, or enzymes described herein.

[0090] Also provided herein is the use of an organic solvent, preferably toluene, to increase the yield of alpha-cyclodextrin obtained in a method for producing alpha-cyclodextrin such as alpha-cyclodextrin obtained from any one of the methods described herein.

[0091] Also provided herein is the use of CaCl2 and / or ethanol to increase the yield of alpha-cyclodextrin compared to beta-cyclodextrin and gamma-cyclodextrin.

[0092] Also provided herein is the use of CaCl2 and / or ethanol to increase the yield of alpha-cyclodextrin obtained in a method for producing alpha-cyclodextrin such as alpha-cyclodextrin obtained from any one of the methods described herein.

[0093] Generally, the term "sequence identity" refers to the exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence between two polynucleotide or polypeptide sequences. Typically, techniques for determining sequence identity involve determining the nucleotide sequence of a polynucleotide and / or the amino acid sequence encoded thereby, and comparing these sequences to a second nucleotide or amino acid sequence. Two or more sequences (polynucleotides or amino acids) can be compared by determining their percent identity. The percent identity of two sequences, whether nucleic acid or amino acid sequences, is the number of exact matches between the two aligned sequences divided by the length of the longer sequence and multiplied by 100. Percent identity can also be determined, for example, by comparing sequence information using sophisticated BLAST computer programs (including version 2.2.9) available from the National Institutes of Health. The BLAST programs are based on the alignment methods of Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 87:2264-2268 (1990), and are as described in Altschul, et al., J. Mol. Biol., 215:403-410 (1990), Karlin And Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5877 (1993), and Altschul et al., Nucleic Acids Res., 25:3389-3402 (1997). This program can be used to determine the percent identity over the full length of the proteins being compared. The default parameters are set, for example, to optimize searches using short query sequences with the blastp program. In this program, a SEG filter can also be used to mask segments of the query sequence determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993).The desired range of sequence identity is approximately 70% to 100%, and integer values in between. Generally, this disclosure encompasses sequences having at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 98% sequence identity with any of the sequences provided herein.

[0094] As used herein, the term "about" generally refers to a range that is 15% more or less than the numerical value described within the context of a particular usage. For example, "about 10" includes the range from 8.5 to 11.5.

[0095] As used herein, the term "or" is used non-exclusively to include "or" and "and". For example, "A or B" includes, unless otherwise stated, "A but not B", "B but not A", and "A and B".

[0096] As used herein, "a", "an", and "the" can include a plurality of referents unless explicitly and specifically limited to one referent. The headings of the sections used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0097] Numbered embodiments The following embodiments enumerate non-limiting permutations of combinations of features disclosed herein. Other permutations of combinations of features are also contemplated. In particular, each of these numbered embodiments is contemplated as being dependent or related to all of the embodiments with a preceding or subsequent number, regardless of the order in which they are described.

[0098] Embodiment 1: A method for producing a composition containing cyclodextrin, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with an enzyme capable of converting amylose to cyclodextrin under conditions that allow the conversion of amylose to cyclodextrin, thereby producing the composition containing cyclodextrin, wherein the enzyme capable of converting amylose to cyclodextrin in (b) is a variant enzyme capable of producing a higher amount and / or concentration of alpha-cyclodextrin than a wild-type enzyme capable of converting amylose to cyclodextrin, the composition containing cyclodextrin contains alpha-cyclodextrin and may further contain, optionally, beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof, and the composition containing cyclodextrin contains a higher amount and / or concentration of alpha-cyclodextrin than beta-cyclodextrin, gamma-cyclodextrin, or both.

[0099] Embodiment 2: The method according to Embodiment 1, wherein the enzyme in (a) is amylosucrase or the enzyme mixture in (a) contains amylosucrase.

[0100] Embodiment 3: The method according to Embodiment 2, wherein the amylosucrase is a variant amylosucrase containing at least one amino acid variant relative to wild-type amylosucrase.

[0101] Embodiment 4: The method according to Embodiment 3, wherein the variant amylosucrase is capable of producing a higher amount and / or concentration of amylose from sucrose relative to wild-type amylosucrase.

[0102] Embodiment 5: The method according to Embodiment 3 or 4, wherein the wild-type amylosucrase is Cellulomonas carboniz T26 amylosucrase.

[0103] Embodiment 6: The method according to Embodiment 5, wherein the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0104] Embodiment 7: The method according to Embodiment 3 or 4, wherein the wild-type amylosucrase is Neisseria polysaccharea amylosucrase.

[0105] Embodiment 8: The method according to Embodiment 7, wherein the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0106] Embodiment 9: The method according to any one of Embodiments 3 to 8, wherein the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0107] Embodiment 10: The method according to any one of Embodiments 3 to 9, wherein the at least one amino acid variant comprises at least one amino acid substitution with respect to the wild-type amylosucrase.

[0108] Embodiment 11: The method according to Embodiment 10, wherein the at least one amino acid substitution comprises an amino acid substitution at position 234 with respect to the wild-type amylosucrase having the amino acid sequence of SEQ ID NO: 2.

[0109] Embodiment 12: The method according to Embodiment 11, wherein the amino acid substitution at position 234 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H.

[0110] Embodiment 13: The method according to embodiment 1, wherein the enzyme mixture in (a) comprises at least two enzymes that can collectively or in combination convert sucrose to amylose.

[0111] Embodiment 14: The method according to embodiment 13, wherein the enzyme mixture comprises sucrose phosphorylase.

[0112] Embodiment 15: The method according to embodiment 14, wherein the sucrose phosphorylase can convert sucrose to glucose-1-phosphate.

[0113] Embodiment 16: The method according to embodiment 15, wherein the contacting in (a) further comprises contacting the sucrose with the sucrose phosphorylase under conditions that allow conversion of the sucrose to glucose-1-phosphate.

[0114] Embodiment 17: The method according to any one of embodiments 14 to 16, wherein the sucrose phosphorylase is selected from the group consisting of Bifidobacterium longum sucrose phosphorylase, Leuconostoc mesenteroides sucrose phosphorylase, and Streptococcus mutans sucrose phosphorylase.

[0115] Embodiment 18: The method according to any one of embodiments 14 to 17, wherein the sucrose phosphorylase comprises, or consists of, an amino acid sequence of any one of SEQ ID NOs: 17 to 20, or an amino acid sequence having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 17 to 20.

[0116] Embodiment 19: The method according to one of embodiments 13 to 18, wherein the enzyme mixture comprises alpha-glucan phosphorylase.

[0117] Embodiment 20: The method according to embodiment 19, wherein the alpha - glucan phosphorylase can convert the glucose - 1 - phosphate into amylose.

[0118] Embodiment 21: The method according to embodiment 20, further comprising contacting the glucose - 1 - phosphate with the alpha - glucan phosphorylase under conditions that enable the conversion of the glucose - 1 - phosphate into amylose.

[0119] Embodiment 22: The method according to any one of embodiments 19 to 21, wherein the alpha - glucan phosphorylase is selected from the group consisting of Solanum tuberosum alpha - glucan phosphorylase, S. tokodaii strain 7 alpha - glucan phosphorylase, and C. callunae DSM 20145 alpha - glucan phosphorylase.

[0120] Embodiment 23: The method according to any one of embodiments 19 to 22, wherein the alpha - glucan phosphorylase comprises, or consists of, an amino acid sequence of any one of SEQ ID NOs: 21 - 24, or an amino acid sequence having at least about 70% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 21 - 24.

[0121] Embodiment 24: The method according to any one of embodiments 1 to 23, wherein the enzyme capable of converting the amylose in (b) into cyclodextrin comprises variant cyclodextrin glucanotransferase.

[0122] Embodiment 25: The method according to embodiment 24, wherein the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild - type cyclodextrin glucanotransferase.

[0123] Embodiment 26: The method according to Embodiment 25, wherein the wild-type cyclodextrin glucanotransferase is Paenibacillus macerans cyclodextrin glucanotransferase.

[0124] Embodiment 27: The method according to Embodiment 26, wherein the wild-type cyclodextrin glucanotransferase comprises, or consists of, any one amino acid sequence of SEQ ID NOs: 25 to 28.

[0125] Embodiment 28: The method according to any one of Embodiments 24 to 27, wherein the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity with any one amino acid sequence of SEQ ID NOs: 25 to 28.

[0126] Embodiment 29: The method according to any one of Embodiments 25 to 28, wherein the at least one amino acid variant comprises at least one amino acid substitution with respect to the wild-type cyclodextrin glucanotransferase.

[0127] Embodiment 30: The method according to Embodiment 29, wherein the at least one amino acid substitution comprises an amino acid substitution at position 146 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28.

[0128] Embodiment 31: The method according to Embodiment 30, wherein the amino acid substitution at position 146 is selected from the group consisting of R146A and R146P.

[0129] Embodiment 32: The method according to Embodiment 29, wherein the at least one amino acid substitution comprises an amino acid substitution at position 147 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28.

[0130] Embodiment 33: The method according to embodiment 32, wherein the amino acid substitution at position 147 is selected from the group consisting of D147P and D147A.

[0131] Embodiment 34: The method according to embodiment 29, wherein the at least one amino acid substitution includes an amino acid substitution at position 146 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28, and an amino acid substitution at position 147 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28.

[0132] Embodiment 35: The method according to embodiment 34, wherein the amino acid substitution at position 146 is selected from the group consisting of R146A and R146P.

[0133] Embodiment 36: The method according to embodiment 34 or 35, wherein the amino acid substitution at position 147 is selected from the group consisting of D147P and D147A.

[0134] Embodiment 37: The method according to embodiment 29, wherein the at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28.

[0135] Embodiment 38: The method according to embodiment 37, wherein the amino acid substitution at position 372 is D372K.

[0136] Embodiment 39: The method according to embodiment 29, wherein the at least one amino acid substitution includes an amino acid substitution at position 89 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28.

[0137] Embodiment 40: The method according to embodiment 39, wherein the amino acid substitution at position 89 is Y89R.

[0138] Embodiment 41: The method according to embodiment 29, wherein the at least one amino acid substitution includes an amino acid substitution at position 372 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28, and an amino acid substitution at position 89 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28.

[0139] Embodiment 42: The method according to embodiment 41, wherein the amino acid substitution at position 372 is D372K.

[0140] Embodiment 43: The method according to embodiment 41 or 42, wherein the amino acid substitution at position 89 is Y89R.

[0141] Embodiment 44: The method according to any one of embodiments 1 to 43, wherein the contacting in (a), the contacting in (b), or both of them further includes adding at least one additive that increases the yield of alpha-cyclodextrin with respect to beta-cyclodextrin, gamma-cyclodextrin, or both during (a), (b), or both.

[0142] Embodiment 45: The method according to embodiment 44, wherein the at least one additive is CaCl2.

[0143] Embodiment 46: The method according to embodiment 45, wherein the CaCl2 is added at a concentration of about 1 mM to about 100 mM.

[0144] Embodiment 47: The method according to any one of embodiments 44 to 46, wherein the at least one additive is ethanol.

[0145] Embodiment 48: The method according to embodiment 47, wherein the ethanol is added at a concentration of about 1 v / v% to about 10 v / v%.

[0146] Embodiment 49: The method according to any one of Embodiments 1 to 48, wherein the contacting in (a) and the contacting in (b) are sequentially performed.

[0147] Embodiment 50: The method according to any one of Embodiments 1 to 48, wherein the contacting in (a) and the contacting in (b) are performed simultaneously or substantially simultaneously.

[0148] Embodiment 51: The method according to any one of Embodiments 1 to 50, wherein the amylose produced in (a) is not purified or isolated before the contacting in (b).

[0149] Embodiment 52: The method according to any one of Embodiments 1 to 51, wherein the contacting in (a), the contacting in (b), or both are performed in vitro.

[0150] Embodiment 53: The method according to Embodiment 52, wherein the contacting in (a), the contacting in (b), or both are performed in a container, vial, bottle, test tube, well, plate, or wrapper.

[0151] Embodiment 54: The method according to Embodiment 52 or 53, wherein at least one enzyme of the enzyme or enzyme mixture in (a), the variant enzyme in (b), or both are purified enzymes, isolated enzymes, or both.

[0152] Embodiment 55: The method according to any one of Embodiments 52 to 54, wherein at least one enzyme of the enzyme or enzyme mixture in (a), the variant enzyme in (b), or both are recombinantly produced enzymes.

[0153] Embodiment 56: The method according to any one of Embodiments 1 to 51, wherein the contacting in (a), the contacting in (b), or both are performed in vivo.

[0154] The method according to embodiment 56, wherein the contacting in (a), the contacting in (b), or both are performed in a recombinant host cell.

[0155] Embodiment 58: The method according to embodiment 57, wherein the recombinant host cell contains a heterologous nucleic acid encoding at least one enzyme of the enzyme or the enzyme mixture in (a), the variant enzyme in (b), or both.

[0156] Embodiment 59: The method according to embodiment 57 or 58, wherein the recombinant host cell is a microbial cell.

[0157] Embodiment 60: The method according to embodiment 59, wherein the microbial cell is a bacterial cell.

[0158] Embodiment 61: The method according to any one of embodiments 1 to 60, wherein the ratio of alpha-cyclodextrin to beta-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

[0159] Embodiment 62: The method according to any one of embodiments 1 to 61, wherein the ratio of alpha-cyclodextrin to gamma-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

Examples

[0160] Example 1. Variant cyclodextrin glucanotransferase can increase the production of alpha-cyclodextrin compared to beta-cyclodextrin and gamma-cyclodextrin. This example demonstrates that cyclodextrin glucanotransferase enzymes were able to increase the production of alpha-cyclodextrin from amylose compared to either, or both, beta-cyclodextrin and gamma-cyclodextrin. In this example, several different cyclodextrin glucanotransferase enzymes ("PMcgt2" having the amino acid sequence according to SEQ ID NO: 28, "PMcgt2[AP]" having the amino acid sequence according to SEQ ID NO: 33, "PMcgt2[PA]" having the amino acid sequence according to SEQ ID NO: 34, "PMcgt2[PP]" having the amino acid sequence according to SEQ ID NO: 35, and "PMcgt2[KR]" having the amino acid sequence according to SEQ ID NO: 41) were expressed in Escherichia coli and then separated from the insoluble cell debris mixture by centrifugation. The cyclodextrin glucanotransferase enzymes were exposed to soluble starch (30 g / L) at 45 °C for 1 hour in 100 mM sodium citrate buffer at pH 6.0. The amounts of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin were measured by HPLC. Figures 4 and Table 6 below demonstrate that only certain mutant cyclodextrin glucanotransferase enzymes (PMcgt2, PMcgt2[AP], PMcgt2[PA], PMcgt2[PP], and PMcgt2[KR]) were able to produce a high ratio of alpha-cyclodextrin relative to gamma-cyclodextrin. Figures 4 and Table 6 further demonstrate that some of the cyclodextrin glucanotransferase enzymes tested (PMcgt2[PA] and PMcgt2[PP]) were also able to produce a high ratio of alpha-cyclodextrin relative to beta-cyclodextrin, and that PMcgt2[PP] functioned best.

Table 6

[0161] Example 2. One-pot synthesis of alpha-cyclodextrin from sucrose In this example, two enzyme systems were used to produce alpha-cyclodextrin from sucrose (i.e., method step (a) was a one-enzyme method (e.g., as described herein), and method step (b) was a one-enzyme method (e.g., as described herein)). Amylosucrase R234Q (having the amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having the amino acid sequence according to SEQ ID NO: 35) were expressed in Escherichia coli and then separated from the cell debris mixture. Next, 200 μL of amylosucrase (SEQ ID NO: 3) and various amounts of cyclodextrin glucanotransferase (SEQ ID NO: 35; 30 μL, 50 μL, and 100 μL) were exposed to 250 g / L sucrose in 0.1 M sodium citrate buffer at 50 °C at different pHs (pH 6.0, pH 7.0, and pH 8.0). The levels of alpha-cyclodextrin were measured by HPLC at various time points (1 hour, 2 hours, and 3 hours). Figure 5 shows that alpha-cyclodextrin can be produced from sucrose under various different pH conditions by a one-pot synthesis reaction.

[0162] Example 3. Various additives can improve the production of alpha-cyclodextrin in the one-pot synthesis reaction. In this example, alpha-cyclodextrin was produced from sucrose using a two-enzyme system (i.e., method step (a) was a one-enzyme method (e.g., as described herein), and method step (b) was a one-enzyme method (e.g., as described herein)). Amylosucrase R234Q (having the amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having the amino acid sequence according to SEQ ID NO: 35) were expressed in Escherichia coli and then separated from the cell debris mixture. Next, 1 mL of amylosucrase (SEQ ID NO: 3) and 1 mL of cyclodextrin glucanotransferase (SEQ ID NO: 35) were exposed to 300 g / L sucrose in 0.1 M sodium citrate buffer at pH 6.5 and 45 °C. Various additives were added to the reaction mixture (10 mM CaCl2, 2% ethanol (v / v), or a combination of 10 mM CaCl2 and 2% ethanol (v / v)). The level of alpha-cyclodextrin was measured by HPLC at various time points (2 h, 3 h, 4 h, and 5 h). Figure 6 demonstrates that the addition of ethanol, or a combination of CaCl2 and ethanol, was able to increase the production of alpha-cyclodextrin from sucrose in a one-pot synthesis reaction. CK refers to a reaction in which neither CaCl2 nor ethanol was added to the reactants. In this reaction, the enzyme processed the conversion of sucrose to product under the base conditions of 0.1 M citrate at pH 6.5.

[0163] Example 4. Lyophilization of amylosucrase cell slurry and cell lysate using a stabilizing compound. The lysate and whole cell slurry of amylosucrase (having the amino acid sequence according to SEQ ID NO: 3) were lyophilized with various stabilizing compounds. More specifically, 0.5 w / v%, 1.0 w / v%, or 5.0 w / v% of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, or beta-cyclodextrin was added to 1 mL of the lysate or cell slurry. The mixture was then lyophilized over a period of 2 days.

[0164] The obtained substance was resuspended in 1 mL of water and dissolved by shaking at 1200 rpm for 30 minutes at room temperature (about 25°C). The enzyme activity retention rate of the obtained solution was measured as described below.

[0165] Enzymatic activity of amylosucrase To 2.67 mL of a solution of sucrose (100 g / L) dissolved in a sodium citrate buffer (0.1 M) at pH 7 and 40°C, 33 μL of an amylosucrase cell-free lysate (or whole cell slurry) was added. The solution was shaken at 1200 rpm for 1 hour. The starch concentration was quantified by spectrophotometric analysis. One activity unit (U / mL) was defined as the amount of enzyme required to produce 1 g / L of amylose per minute at pH 7 and 40°C. The enzyme activity of the non-lyophilized cell lysate (or whole cell slurry respectively) was compared. The results are shown in Figures 7A and 7B respectively.

[0166] As shown in Figure 7, when a stabilizing compound is added to amylosucrase before lyophilization, the stability of the enzyme is improved. In particular, it has been demonstrated that the addition of 5% sucrose, 0.5% mannitol, and 0.5% sorbitol improves the enzyme activity retention rate of the amylosucrase cell lysate and whole cell slurry.

[0167] The examples are presented for the purpose of illustrating various embodiments of the present invention and are not intended to limit the present invention in any way. These examples, in conjunction with the methods described herein, represent the preferred embodiments at the present time, are exemplary, and are not intended to limit the scope of the present invention. Modifications and other uses within the spirit of the present invention as defined by the claims will be apparent to those skilled in the art.

[0168] Preferred embodiments of the present invention have been shown and described herein, but it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Without departing from the present invention, numerous variations, modifications, and substitutions will immediately occur to those skilled in the art. In practicing the present invention, it is to be understood that various alternative means to the embodiments of the present invention described herein may be used. It is intended that the following claims define the scope of the present invention and that methods and structures within the scope of these claims and their equivalents be thereby covered.

Claims

1. A method for producing a composition comprising cyclodextrin, the method comprising: (a) contacting the sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with an enzyme capable of converting amylose to cyclodextrin under conditions that allow the conversion of amylose to cyclodextrin, thereby producing the composition comprising cyclodextrin, wherein the enzyme capable of converting amylose to cyclodextrin in (b) is a variant enzyme capable of producing alpha-cyclodextrin in an amount and / or concentration higher than that of wild-type enzyme capable of converting amylose to cyclodextrin, beta-cyclodextrin, gamma-cyclodextrin, or both; the composition comprising cyclodextrin comprises alpha-cyclodextrin and may further comprise beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof, as appropriate; the composition comprising cyclodextrin comprises alpha-cyclodextrin in an amount and / or concentration higher than that of beta-cyclodextrin, gamma-cyclodextrin, or both, said method.

2. The method according to claim 1, wherein the enzyme in (a) is amylosucrase or the enzyme mixture in (a) comprises amylosucrase.

3. The amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to wild-type amylosucrase, for example, the variant amylosucrase is capable of producing a higher amount and / or concentration of amylose from sucrose relative to wild-type amylosucrase, the method according to claim 2.

4. The method according to claim 3, (i) wherein the wild-type amylosucrase is Cellulomonas carbonis T26 amylosucrase, for example, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 1, or (ii) the wild-type amylosucrase is Neisseria polysaccharea amylosucrase, for example, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 2, the method.

5. The method according to any one of claims 3 to 4, wherein the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO:

2.

6. The method according to any one of claims 3 to 5, wherein the at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type amylosucrase, preferably, the at least one amino acid substitution comprises an amino acid substitution at position 234 of the amino acid sequence of the wild-type amylosucrase having the amino acid sequence of SEQ ID NO: 2, preferably, the amino acid substitution at position 234 is selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H, preferably, the amino acid substitution at position 234 is R234Q, the method.

7. The method according to claim 1, wherein the enzyme mixture of (a) comprises at least two enzymes capable of converting sucrose to amylose collectively or in combination.

8. The method according to claim 7, wherein the enzyme mixture comprises sucrose phosphorylase, preferably, the sucrose phosphorylase is capable of converting sucrose to glucose-1-phosphate, preferably, the contacting in (a) further comprises contacting the sucrose with the sucrose phosphorylase under conditions that allow conversion of the sucrose to glucose-1-phosphate, the method.

9. The method according to claim 8, wherein the sucrose phosphorylase is selected from the group consisting of Bifidobacterium longum sucrose phosphorylase, Leuconostoc mesenteroides sucrose phosphorylase, and Streptococcus mutans sucrose phosphorylase.

10. The method according to any one of claims 8 to 9, wherein the sucrose phosphorylase comprises an amino acid sequence of any one of SEQ ID NOs: 17 to 20, or an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 17 to 20.

11. The method according to any one of claims 7 to 10, wherein the enzyme mixture comprises alpha-glucan phosphorylase, preferably, the alpha-glucan phosphorylase is capable of converting the glucose-1-phosphate to amylose, preferably, the contacting in (a) further comprises contacting the glucose-1-phosphate with the alpha-glucan phosphorylase under conditions that allow conversion of the glucose-1-phosphate to amylose. The method.

12. The method according to claim 11, wherein the alpha-glucan phosphorylase is selected from the group consisting of Solanum tuberosum alpha-glucan phosphorylase, S. tokodaii strain 7 alpha-glucan phosphorylase, and C. callunae DSM 20145 alpha-glucan phosphorylase.

13. The method according to any one of claims 11 to 12, wherein the alpha-glucan phosphorylase comprises an amino acid sequence of any one of SEQ ID NOs: 21 to 24, or an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 21 to 24.

14. The method according to any one of claims 1 to 13, wherein the enzyme capable of converting the amylose in (b) to cyclodextrin comprises variant cyclodextrin glucanotransferase.

15. The method according to claim 14, wherein the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type cyclodextrin glucanotransferase.

16. The wild-type cyclodextrin glucanotransferase is Paenibacillus macerans cyclodextrin glucanotransferase, and preferably, the wild-type cyclodextrin glucanotransferase comprises, or consists of, any one amino acid sequence of SEQ ID NOs: 25 to 28, the method according to claim 15.

17. The variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity with any one amino acid sequence of SEQ ID NOs: 25 to 28, the method according to any one of claims 14 to 16.

18. The method according to any one of claims 14 to 17, wherein the at least one amino acid variant comprises at least one amino acid substitution with respect to the wild-type cyclodextrin glucanotransferase, preferably, (i) the at least one amino acid substitution comprises an amino acid substitution at position 146 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28, preferably, the amino acid substitution at position 146 is selected from the group consisting of R146A and R146P, or (ii) the at least one amino acid substitution comprises an amino acid substitution at position 147 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28, preferably, the amino acid substitution at position 147 is selected from the group consisting of D147P and D147A, or (iii) the at least one amino acid substitution comprises an amino acid substitution at position 146 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28, and an amino acid substitution at position 147 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 28, preferably, the amino acid substitution at position 146 is selected from the group consisting of R146A and R146P, and / or, preferably, the amino acid substitution at position 147 is selected from the group consisting of D147P and D147A, or (iv) the at least one amino acid substitution includes an amino acid substitution at position 372 of a wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28, preferably, the amino acid substitution at position 372 is D372K, or (v) the at least one amino acid substitution includes an amino acid substitution at position 89 of a wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28, preferably, the amino acid substitution at position 89 is Y89R, or (vi) the at least one amino acid substitution includes an amino acid substitution at position 372 of a wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28 and an amino acid substitution at position 89 of a wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26 or 28, preferably, the amino acid substitution at position 372 is D372K, and / or preferably, the amino acid substitution at position 89 is Y89R, the method.

19. The method according to any one of claims 1 to 18, wherein the contacting in (a), the contacting in (b), or both of them further includes adding at least one additive that increases the yield of alpha-cyclodextrin with respect to beta-cyclodextrin, gamma-cyclodextrin, or both during (a), (b), or both.

20. The method according to claim 19, (i) the at least one additive is CaCl ２ and Preferably, the CaCl ２ is added at a concentration of about 1 mM to about 100 mM, and / or (ii) the at least one additive is ethanol, preferably, the ethanol is added at a concentration of about 1 v / v% to about 10 v / v%, the method.

21. The method according to any one of claims 1 to 20, wherein the contacting in (a) and the contacting in (b) are performed sequentially, or the contacting in (a) and the contacting in (b) are performed simultaneously or substantially simultaneously.

22. The method according to any one of claims 1 to 21, wherein the amylose produced in (a) is not purified or isolated before the contacting in (b).

23. The method according to any one of claims 1 to 22, wherein the contacting in (a), the contacting in (b), or both are performed in vitro.

24. The method according to claim 23, wherein the contacting in (a), the contacting in (b), or both are performed in a container, vial, bottle, test tube, well, plate, or enclosure.

25. The method according to claim 23 or 24, wherein at least one enzyme of the enzyme or the enzyme mixture in (a), the variant enzyme in (b), or both are purified enzymes, isolated enzymes, or both. Preferably, the method, wherein at least one enzyme of the enzyme or the enzyme mixture in (a), the variant enzyme in (b), or both are recombinantly produced enzymes.

26. The method according to any one of claims 1 to 22, wherein the contacting in (a), the contacting in (b), or both are performed in vivo.

27. The method according to claim 26, wherein the contacting in (a), the contacting in (b), or both are performed in a recombinant host cell. Preferably, the recombinant host cell contains a heterologous nucleic acid encoding at least one enzyme of the enzyme or the enzyme mixture in (a), the variant enzyme in (b), or both. Preferably, the recombinant host cell is a microbial cell. Preferably, the method, wherein the microbial cell is a bacterial cell.

28. The method according to any one of claims 1 to 27, wherein the ratio of alpha-cyclodextrin to beta-cyclodextrin in the composition containing cyclodextrin is at least 2:

1.

29. The method according to any one of claims 1 to 28, wherein the ratio of alpha-cyclodextrin to gamma-cyclodextrin in the composition containing cyclodextrin is at least 2:1, preferably at least 100:

1.

30. The method according to any one of claims 1 to 29, wherein at least one enzyme of the enzyme or the enzyme mixture in (a), the variant enzyme in (b), or both are produced in Pichia yeast cells.

31. The method according to any one of claims 1 to 30, wherein the contacting in (a) and / or the contacting in (b) is carried out in a reaction mixture containing an organic solvent, preferably toluene.

32. The method according to any one of claims 1 to 31, wherein at least one enzyme of the enzyme or the enzyme mixture in (a), the variant enzyme in (b), or both are immobilized on a resin.

33. The method according to any one of claims 1 to 32, wherein at least one of the enzyme or the enzyme mixture in (a), the variant enzyme in (b), or both are provided in a cell slurry or a whole cell lysate.

34. The method according to claim 33, wherein the cell slurry or the whole cell lysate further contains a stabilizing compound selected from the group consisting of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, and combinations thereof, preferably, the stabilizing compound is selected from the group consisting of mannitol, sorbitol, sucrose, and combinations thereof, the method.

35. The ratio of alpha-cyclodextrin in the composition to beta-cyclodextrin, gamma-cyclodextrin, or both is at least 2:1, steps (a) and (b) are carried out simultaneously, steps (a) and (b) are carried out at about 45 °C to about 55 °C, steps (a) and (b) are carried out at a pH of about 7.0 to about 7.5, steps (a) and (b) are carried out in a reaction mixture containing water, ethanol, CaCl ２ , and, as appropriate, an organic solvent (preferably toluene), and the entire reaction is carried out over 8 hours or less, the method according to any one of claims 1 to 34.

36. A composition containing cyclodextrin, wherein the cyclodextrin contains alpha-cyclodextrin and may further contain beta-cyclodextrin, gamma-cyclodextrin, or any combination thereof as appropriate, and the composition containing cyclodextrin contains alpha-cyclodextrin in an amount and / or concentration higher than that of beta-cyclodextrin, gamma-cyclodextrin, or both, the composition obtained from the method according to any one of claims 1 to 35.

37. The composition according to claim 36, wherein the ratio of alpha-cyclodextrin to beta-cyclodextrin in the composition containing cyclodextrin is at least 2:1, preferably at least 100:1, and / or the ratio of alpha-cyclodextrin to gamma-cyclodextrin in the composition containing cyclodextrin is at least 2:1, preferably at least 100:1, the composition.

38. The composition according to claim 36 or 37, wherein the yield percentage of alpha-cyclodextrin is at least about 10%, preferably at least about 60%.

39. Use of sucrose for the production of alpha-cyclodextrin, wherein the production method is the method according to any one of claims 1 to 35.

40. Use of one or more of the enzymes of SEQ ID NO: 1 to SEQ ID NO: 42 for the production of alpha-cyclodextrin, preferably, the production method is the method according to any one of claims 1 to 35.

41. An enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NO: 1 to 42, or an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with any one of the amino acid sequences of SEQ ID NO: 1 to 42.

42. The enzyme according to claim 41, (i) a variant amylosucrase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NO: 3 to 16 or 42, (ii) a variant sucrose phosphorylase enzyme comprising or consisting of the amino acid sequence of SEQ ID NO: 20, (iii) a variant alpha-glucan phosphorylase enzyme comprising or consisting of the amino acid sequence of SEQ ID NO: 24, or (iv) a variant cyclodextrin glucanotransferase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NO: 29 to 41 is the said enzyme.

Images