Method for producing beta-cyclodextrin

Abstract

Provided herein is a method for the enzymatic production of beta-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 beta-cyclodextrin. Optionally, the method produces beta-cyclodextrin in a high yield compared to alpha-cyclodextrin, gamma-cyclodextrin, or both.

Description

Background Art

[0001] Cyclodextrin is a type 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 variability, purification, and the cost of 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. The present disclosure meets this unmet need by providing a method for biosynthetically producing cyclodextrin that does not use starch as a starting material. 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 cyclodextrin product, more desirable purity, and a decrease in the amount of byproduct waste, as well as byproducts 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 that can produce a higher amount and / or concentration of beta-cyclodextrin than a wild-type enzyme capable of converting amylose to cyclodextrin, and the composition comprising cyclodextrin comprises beta-cyclodextrin and may further comprise alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof, and the composition comprising cyclodextrin comprises a higher amount and / or concentration of beta-cyclodextrin than alpha-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 can produce 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 the amino acid sequence of SEQ ID NO: 1. Optionally, the wild-type amylosucrase is Neisseria polysaccharea amylosucrase. Optionally, the wild-type amylosucrase comprises 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 of the amino acid sequence of 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 capable of converting sucrose to amylose, either in combination or collectively. In some cases, the enzyme mixture comprises sucrose phosphorylase. In some cases, sucrose phosphorylase is capable of converting 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, preferably at least about 90% 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 is capable of converting 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 of 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, preferably at least about 90% 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 Bacillus sp. strain number 38-2 cyclodextrin glucanotransferase. In some cases, Bacillus sp. strain number 38-2 cyclodextrin glucanotransferase comprises, or consists of, the amino acid sequence of SEQ ID NO: 25. In some cases, the wild-type cyclodextrin glucanotransferase is Bacillus circulans strain 251 cyclodextrin glucanotransferase. In some cases, Bacillus circulans strain 251 cyclodextrin glucanotransferase comprises, or consists of, the amino acid sequence of SEQ ID NO: 26 or 27, such as SEQ ID NO: 27. In some cases, the variant cyclodextrin glucanotransferase comprises, or consists 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: 25-27.In some cases, at least one amino acid variant comprises at least one amino acid substitution relative to wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution comprises an amino acid substitution at amino acid position 31 relative to wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 27. In some cases, the amino acid substitution at amino acid position 31 is selected from the group consisting of A31R, A31P, and A31T. In some cases, the contacting in (a) and the contacting in (b) are performed sequentially. In some cases, the contacting in (a) and the contacting in (b) are performed simultaneously or substantially simultaneously. In some cases, the amylose produced in (a) is not purified or isolated prior to the contacting in (b). In some cases, the contacting in (a), the contacting in (b), or both are performed in vitro. In some cases, the contacting in (a), the contacting in (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 in (a), the variant enzyme in (b), or both are purified enzyme, isolated enzyme, or both. In some cases, at least one enzyme of the enzyme or enzyme mixture in (a), the variant enzyme in (b), or both are recombinantly produced enzymes. In some cases, the contacting in (a), the contacting in (b), or both are performed in vivo. In some cases, the contacting in (a), the contacting in (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 in (a), the variant enzyme in (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 beta-cyclodextrin to alpha-cyclodextrin in the composition containing cyclodextrin is at least 2:1. In some cases, the ratio of beta-cyclodextrin to gamma-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

[0004] The present invention also provides a method for purifying beta-cyclodextrin, the method comprising i. preparing a crude composition containing beta-cyclodextrin; ii. obtaining a first precipitate containing beta-cyclodextrin from the crude composition; iii. dissolving the first precipitate to obtain a first solution containing beta-cyclodextrin; iv. filtering the first solution to obtain a second solution containing beta-cyclodextrin; v. crystallizing and / or precipitating the second solution to obtain a purified beta-cyclodextrin composition. Accordingly, the present invention also provides a purified beta-cyclodextrin composition. In some cases, step ii includes filtering the crude composition, subjecting the crude composition to centrifugation, subjecting the crude composition to a sedimentation operation, and / or washing with water to obtain the first precipitate. In some cases, step iii includes dissolving the precipitate in water.

[0005] Incorporation by reference All published documents, patents, and patent applications described herein are incorporated herein by reference to the same extent as if each individual published document, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0006] The novel features of the present invention are particularly pointed out 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

[0007]

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Mode for Carrying Out the Invention

[0008] Current cyclodextrin production methods have issues such as supply chain shortages and problems 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 cyclodextrin from sucrose as a starting material.

[0009] Provided herein are methods for producing a composition comprising cyclodextrin. Also provided herein are methods for the enzymatic synthesis of beta-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. Provided herein are methods for the enzymatic conversion of sucrose to beta-cyclodextrin using various enzymes. The method generally involves 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 involves the enzymatic conversion of amylose to beta-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).

[0010] Cyclodextrin is formed by the cyclic arrangement of glucopyranose units conjugated by α-1,4 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 supervision of the European Medicines Agency (EMA) in Europe and the Food and Drug Administration (FDA) in the United States. Since natural CD can be ingested without significant absorption, it has received "safe food certification" from the FDA and is generally regarded as a molecule with "GRAS status".

[0011] Beta-cyclodextrin is widely used in the pharmaceutical industry. Various derivatives of beta-cyclodextrin have been produced to improve the oral bioavailability and solubility of cyclodextrin. For example, modifying the hydroxyl groups of cyclodextrin with hydroxypropyl groups dramatically improves the solubility of cyclodextrin. Some of the possible derivatives include randomly methylated beta-cyclodextrin and branched beta-cyclodextrin.

[0012] 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 beta-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, alpha-cyclodextrin, gamma-cyclodextrin, or both. Optionally, the composition comprising cyclodextrin comprises beta-cyclodextrin and may further comprise, optionally, alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof. Optionally, the composition comprising cyclodextrin comprises beta-cyclodextrin in an amount and / or concentration (e.g., weight %, mol %, or w / v) higher than that of alpha-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).

[0013] Method step (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 alpha-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 to convert sucrose to amylose. In alternative aspects, the method includes the use of an enzyme mixture (e.g., two enzymes) that collectively or combinatorially 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.

[0014] One - enzyme method for producing amylose from sucrose In some aspects, the enzyme is amylosucrase. FIG. 2A shows a schematic of a single-enzyme method for producing amylose from sucrose. In this example, sucrose is contacted with amylosucrase, which converts the sucrose to amylose. Optionally, the amylosucrase is wild-type amylosucrase. For example, the wild-type amylosucrase can be Cellulomonas carboniz T26 amylosucrase (NCBI accession number N868_11335). Optionally, the wild-type Cellulomonas carboniz T26 amylosucrase can have the amino acid sequence of SEQ ID NO: 1. Optionally, the wild-type amylosucrase can be Neisseria polysaccharea amylosucrase (NCBI accession number AJ011781). Optionally, the wild-type Neisseria polysaccharea amylosucrase can have the amino acid sequence of 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

[0015] In some embodiments, the amylosucrase is a variant amylosucrase that comprises at least one amino acid variant relative to wild-type amylosucrase. The variant amylosucrase can include one or more amino acid substitutions, deletions, insertions, and / or modifications relative to wild-type amylosucrase. Optionally, the variant amylosucrase can produce a higher amount and / or concentration of amylose from sucrose relative to wild-type amylosucrase.

[0016] In some cases, the variant amylosucrase comprises at least one amino acid variant relative to wild-type Cellulomonas carboniz T26 amylosucrase (SEQ ID NO: 1). In some cases, the variant amylosucrase comprises at least one amino acid variant relative to wild-type Neisseria polysaccharea amylosucrase (SEQ ID NO: 2). In some cases, 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 wild-type Cellulomonas carboniz T26 amylosucrase, preferably at least about 90% sequence identity. In some cases, 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: 1, preferably at least about 90% sequence identity.In some cases, 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 wild-type Neisseria polysaccharea amylosucrase, preferably at least about 90% sequence identity. In some cases, 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.

[0017] In some cases, at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type amylosucrase. In some cases, at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type Cellulomonas carboniz T26 amylosucrase. In some cases, at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type Neisseria polysaccharea amylosucrase. In some cases, at least one amino acid substitution comprises or consists of an amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2. In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of 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 amino acid position 234 relative to the amino acid sequence of 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 amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is substituted with glutamine (Q), and so on. In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234Q (e.g., SEQ ID NO: 3 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234G (e.g., SEQ ID NO: 4 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234A (e.g., SEQ ID NO: 5 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of SEQ ID NO: 2 is R234S (e.g., SEQ ID NO: 6 in Table 2). In some cases, the amino acid substitution at amino acid position 234 relative to the amino acid sequence of 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 with respect to the amino acid sequence of SEQ ID NO: 2 is R234C (e.g., SEQ ID NO: 8 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234K (e.g., SEQ ID NO: 9 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234I (e.g., SEQ ID NO: 10 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234D (e.g., SEQ ID NO: 11 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234Y (e.g., SEQ ID NO: 12 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234W (e.g., SEQ ID NO: 13 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234E (e.g., SEQ ID NO: 14 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234L (e.g., SEQ ID NO: 15 in Table 2). In some cases, the amino acid substitution at position 234 with respect to the amino acid sequence of SEQ ID NO: 2 is R234H (e.g., SEQ ID NO: 16 in Table 2). In some embodiments, the variant amylosucrase 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 any one of SEQ ID NOs: 3 - 16 or 48 shown in Table 2, or the amino acid sequence according to any one of SEQ ID NOs: 3 - 16 or 48 shown in Table 2.In a preferred embodiment, the variant amylase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 3 to 9 or 48 shown in Table 2.

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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 an 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 an 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 an 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 the 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 the 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 the 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 amino acid substitution R234L 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 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 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 microbial cells. In some embodiments, the amylosucrase is expressed within recombinant host cells (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 comprises contacting sucrose with an enzyme mixture capable of converting sucrose to amylose under conditions that enable the conversion of sucrose to amylose, thereby producing amylose. Optionally, the method comprises contacting sucrose with an enzyme mixture comprising at least two enzymes capable of collectively or combinatorially converting sucrose to amylose. For example, the enzyme mixture can comprise at least sucrose phosphorylase and alpha-glucan phosphorylase. The method can comprise contacting sucrose with at least two enzymes simultaneously or substantially simultaneously. Alternatively, the method can comprise contacting sucrose with at least two enzymes sequentially. Figure 2B shows a schematic of a two-enzyme process 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 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 of amino acid for SEQ ID NO: 19 is Q144R. In some cases, the amino acid substitution at position 155 of amino acid for SEQ ID NO: 19 is N155S. In some cases, the amino acid substitution at position 249 of amino acid 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 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., 75%, at least about 80%, at least about 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., 75%, at least about 80%, at least about 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, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 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, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 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 Streptococcus mutans 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., 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, the sucrose phosphorylase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., 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 includes the amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G relative to SEQ ID NO: 19.

[0023] In some embodiments, the sucrose phosphorylase is derived from a microbial cell. In some cases, the 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, the sucrose phosphorylase is derived from Bifidobacterium longum. In some embodiments, the sucrose phosphorylase is derived from Leuconostoc mesenteroides. In some embodiments, the sucrose phosphorylase is derived from Streptococcus mutans. In some embodiments, the sucrose phosphorylase can be produced within a microbial cell. In some embodiments, the sucrose phosphorylase is expressed within a recombinant host cell (e.g., from a recombinant polynucleotide). In some cases, the sucrose phosphorylase is recombinantly produced. In some cases, the sucrose phosphorylase is produced in a yeast cell (e.g., recombinantly produced). 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 an amino acid substitution 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 comprises or consists of 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: 24, preferably at least about 90% sequence identity, and comprises the amino acid substitutions F39L, N135S, and T706I with respect 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 within recombinant host cells (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 step (b) for the enzymatic conversion of amylose to beta - 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 beta-cyclodextrin. Optionally, the method comprises contacting amylose with an enzyme or enzyme mixture (e.g., two or more enzymes) capable of converting amylose to cyclodextrin under conditions that allow for the conversion of amylose to cyclodextrin. Optionally, the enzyme capable of converting amylose to cyclodextrin is a variant enzyme that can produce a higher amount and / or concentration of beta-cyclodextrin than alpha-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 beta-cyclodextrin using cyclodextrin glucanotransferase. Preferably, the cyclodextrin glucanotransferase produces beta-cyclodextrin from amylose in a higher amount and / or concentration than the amount and / or concentration of alpha-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 may include one or more amino acid substitutions, deletions, insertions, and / or modifications relative to the wild-type cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase can produce a higher amount and / or concentration of beta-cyclodextrin from amylose compared to alpha-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 the wild-type Bacillus sp. (strain number 38-2) cyclodextrin glucanotransferase (e.g., NCBI accession number M19880.1; SEQ ID NO: 25). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to the wild-type B. circulans strain 251 cyclodextrin glucanotransferase (e.g., NCBI accession number X78145.1; SEQ ID NO: 26 or 27). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to the wild-type B. circulans strain 251 cyclodextrin glucanotransferase of SEQ ID NO: 27. 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: 25, 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 SEQ ID NO: 26 or 27, 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 SEQ ID NO: 27, preferably at least about 90% sequence identity.

[0031] In some cases, at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution comprises an amino acid substitution at position 31 relative to the amino acid sequence of SEQ ID NO: 27. In some cases, the amino acid substitution at position 31 relative to the amino acid sequence of SEQ ID NO: 27 is A31R (e.g., SEQ ID NO: 28 in Table 5). In some cases, the amino acid substitution at position 31 relative to the amino acid sequence of SEQ ID NO: 27 is A31P (e.g., SEQ ID NO: 29 in Table 5). In some cases, the amino acid substitution at position 31 relative to the amino acid sequence of SEQ ID NO: 27 is A31T (e.g., SEQ ID NO: 30 in Table 5). In some embodiments, the cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 25 - 30 shown in Table 5.

[0032] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to the wild-type Paenibacillus macerans cyclodextrin glucanotransferase (e.g., NCBI accession number AAA22298.1 or X59045.1; e.g., SEQ ID NOs: 31-34). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to any one of SEQ ID NOs: 31-34. 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) to the amino acid sequence of the 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) to the amino acid sequence of any one of SEQ ID NOs: 31-34, preferably at least about 90% sequence identity.

[0033] 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 position 146 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146A (e.g., SEQ ID NO: 35 in Table 5). In some cases, the amino acid substitution at position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146P (e.g., SEQ ID NO: 36 in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at position 147 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147A (e.g., SEQ ID NO: 37 in Table 5). In some cases, the amino acid substitution at position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 38 in Table 5). In some cases, at least one amino acid substitution includes amino acid substitutions at positions 146 and 147 relative to the amino acid sequence of SEQ ID NO: 34. In some cases, the amino acid substitution at position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146A, and the amino acid substitution at position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 39 in Table 5). In some cases, the amino acid substitution at position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146P, and the amino acid substitution at position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147A (e.g., SEQ ID NO: 40 in Table 5). In some cases, the amino acid substitution at position 146 relative to the amino acid sequence of SEQ ID NO: 34 is R146P, and the amino acid substitution at position 147 relative to the amino acid sequence of SEQ ID NO: 34 is D147P (e.g., SEQ ID NO: 41 in Table 5).

[0034] 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: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is D372K (for example, SEQ ID NO: 42 (for SEQ ID NO: 32) and SEQ ID NO: 45 (for SEQ ID NO: 34) 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: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34 is Y89R (for example, SEQ ID NO: 43 (for SEQ ID NO: 32) and SEQ ID NO: 47 (for SEQ ID NO: 34) 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: 32 or SEQ ID NO: 34 and an amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 32 or SEQ ID NO: 34. In some cases, the amino acid substitution at position 372 with respect to the amino acid sequence of SEQ ID NO: 32 or 34 is D372K, and the amino acid substitution at position 89 with respect to the amino acid sequence of SEQ ID NO: 32 or 34 is Y89R (for example, SEQ ID NO: 44 (for SEQ ID NO: 32) and SEQ ID NO: 47 (for SEQ ID NO: 34) in Table 5).

[0035] In some embodiments, the cyclodextrin glucanotransferase comprises or consists of an amino acid sequence according to any one of SEQ ID NOs: 31 to 47 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: 31 to 47 shown in Table 5.

[0036] 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: 34.

[0037] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 39, 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: 39, preferably at least about 90% sequence identity.

[0038] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 40, 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: 40, preferably at least about 90% sequence identity.

[0039] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 41, 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: 41, preferably at least about 90% sequence identity.

[0040] In another specific embodiment, the cyclodextrin glucanotransferase comprises or consists of the amino acid sequence according to SEQ ID NO: 47, 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: 47, preferably at least about 90% sequence identity.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

Table 5-5

[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: 25, preferably at least about 90% sequence identity. 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 27, preferably at least about 90% sequence identity.

[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: 27, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 31 relative to SEQ ID NO: 27. 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: 27, preferably at least about 90% sequence identity, and an amino acid substitution A31R at amino acid position 31 relative to SEQ ID NO: 27. 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: 27, preferably at least about 90% sequence identity, and an amino acid substitution A31P at amino acid position 31 relative to SEQ ID NO: 27.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: 27, preferably at least about 90% sequence identity, and the amino acid substitution A31T relative to SEQ ID NO: 27.

[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: 34, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, and an amino acid substitution R146A relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, and an amino acid substitution R146P relative to SEQ ID NO: 34.

[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: 34, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, and the amino acid substitution D147P relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, and the amino acid substitution D147A relative to SEQ ID NO: 34.

[0045] 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: 34, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 146 relative to SEQ ID NO: 34, and an amino acid substitution at amino acid position 147 relative to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, the amino acid substitution R146A relative to SEQ ID NO: 34, and the amino acid substitution D147P relative to SEQ ID NO: 34.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: 34, preferably at least about 90% sequence identity, an amino acid substitution R146P with respect to SEQ ID NO: 34, and an amino acid substitution D147A with respect to SEQ ID NO: 34. 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: 34, preferably at least about 90% sequence identity, an amino acid substitution R146P with respect to SEQ ID NO: 34, and an amino acid substitution D147P with respect to SEQ ID NO: 34.

[0046] 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: 32 or 34, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 32 or 34. 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: 32 or 34, preferably at least about 90% sequence identity, and an amino acid substitution D372K relative to SEQ ID NO: 32 or 34.

[0047] 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: 32 or 34, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 32 or 34. 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: 32 or 34, preferably at least about 90% sequence identity, and an amino acid substitution Y89R relative to SEQ ID NO: 32 or 34.

[0048] 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: 32 or 34, preferably at least about 90% sequence identity, an amino acid substitution at amino acid position 372 relative to SEQ ID NO: 32 or 34, and an amino acid substitution at amino acid position 89 relative to SEQ ID NO: 32 or 34. 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: 32 or 34, preferably at least about 90% sequence identity, the amino acid substitution D372K relative to SEQ ID NO: 32 or 34, and the amino acid substitution Y89R relative to SEQ ID NO: 32 or 34.

[0049] In some embodiments, cyclodextrin glucanotransferase is derived from microbial cells. Optionally, cyclodextrin glucanotransferase is isolated and / or purified from microbial cells. Optionally, the microbial cells are bacterial cells. Optionally, the bacterial cells are Escherichia coli. In some embodiments, cyclodextrin glucanotransferase is derived from Bacillus sp. (strain number 38-2). In some embodiments, cyclodextrin glucanotransferase is derived from B. circulans strain 251. 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). Optionally, cyclodextrin glucanotransferase is produced recombinantly. Optionally, cyclodextrin glucanotransferase is produced in yeast cells (e.g., produced recombinantly). Optionally, the yeast cells are Pichia yeast cells such as Pichia pastoris cells.

[0050] In various embodiments, the methods provided herein produce beta-cyclodextrin at a higher ratio than alpha-cyclodextrin, gamma-cyclodextrin, or both. For example, in some cases, the methods provided herein provide a ratio of beta-cyclodextrin to alpha-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 a preferred embodiment, the methods provided herein provide a ratio of beta-cyclodextrin to alpha-cyclodextrin of at least 10:1. For example, the ratio can be 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 a preferred embodiment, the methods provided herein provide a ratio of beta-cyclodextrin to gamma-cyclodextrin of at least 5:1. For example, the ratio can be 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 a preferred embodiment, the methods provided herein provide a ratio of beta-cyclodextrin to both alpha-cyclodextrin and gamma-cyclodextrin of at least 3.5:1. For example, the ratio can be 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 60:1, at least 70:1, at least 80:1, at least 90:1, at least 100:1, or more.

[0051] Throughout the present disclosure, methods are outlined for achieving robust enzyme activity at each step to obtain beta-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, and subsequently, a second enzymatic step of converting amylose to beta-cyclodextrin (e.g., as described herein) is carried out over a second period, thereby enabling a catalytic conversion of amylose to beta-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 beta-cyclodextrin) are carried out in the same reservoir (e.g., one-pot synthesis method).

[0052] 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 beta-cyclodextrin).In some embodiments, the enzyme used in the first enzyme reaction step (e.g., to convert sucrose to amylose as described herein) 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 beta-cyclodextrin).

[0053] 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.

[0054] In some embodiments, the reaction time is an important consideration for obtaining beta-cyclodextrin with maximum yield. In some embodiments, the production of beta-cyclodextrin can involve the decomposition of the product into glucose, maltose, and other sugars. Therefore, it is important to obtain beta-cyclodextrin without decomposition. 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.

[0055] Temperature is an important consideration for maximizing the yield of beta - 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.

[0056] 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.

[0057] In one-pot synthesis, even though the optimal temperatures of the individual enzymes may vary slightly, it is considered that the functionality of the enzyme mixture(s) should be maximized.

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

[0059] 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.

[0060] 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 from about 100 to about 200 rpm, such as about 160 rpm.

[0061] The pH of the reaction mixture can be an important consideration for maximizing the yield of beta-cyclodextrin. In some embodiments, one or more of the enzyme reactions are carried out at a pH of from about 6 to about 8, for example, the pH can be from about 6.5 to about 7.5. In preferred embodiments, one or more of the enzyme reactions are carried out at a pH of from about 7.0 to about 7.5. Preferably, step (a) is carried out at a pH of from about 7.0 to about 7.5. Preferably, step (b) is carried out at a pH of from about 7.0 to about 7.5. Steps (a) and (b) may be carried out at different pHs, but preferably, steps (a) and (b) are carried out at substantially the same pH.

[0062] In some embodiments, one or more of the enzymatic 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 reaction mixture at a concentration of about 50 mM to about 200 mM, such as about 100 mM.

[0063] 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 beta-cyclodextrin obtained from the enzymatic reaction. For example, the addition of an organic solvent can increase the yield of beta-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%, or such as at least about 400% compared to the yield obtained from the enzymatic reaction carried out without the organic solvent. It is believed that the addition of the organic solvent decreases the solubility of beta-cyclodextrin in the reaction mixture, causing beta-cyclodextrin to precipitate and reducing the concentration of beta-cyclodextrin in the reaction mixture, thereby increasing the yield of beta-cyclodextrin. This prevents the degradation of beta-cyclodextrin by the enzyme.

[0064] 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%.

[0065] 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).

[0066] In some embodiments, the enzyme used in step (a) is amylosucrase. In some embodiments, the starting concentration of amylosucrase in the reaction mixture is from about 1 to about 30 U / mL, such as from about 5 to about 25 U / mL, such as from about 8 to about 25 U / mL.

[0067] In some embodiments, the enzyme mixture used in step (a) comprises sucrose phosphorylase and alpha - glucan phosphorylase. In some embodiments, the starting concentration of sucrose phosphorylase in the reaction mixture is from about 1 to about 30 U / mL, such as from about 5 to about 25 U / mL, such as from about 8 to about 25 U / mL. In some embodiments, the starting concentration of alpha - glucan phosphorylase in the reaction mixture is from about 1 to about 30 U / mL, such as from about 5 to about 25 U / mL, such as from about 8 to about 25 U / mL.

[0068] In some embodiments, the enzyme is provided in a whole - cell lysate, and 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 from about 1:1 to about 50:1, such as from about 2:1 to about 50:1, such as from about 5:1 to about 40:1, such as from about 10:1 to about 30:1. In a preferred embodiment, the ratio is about 20:1.

[0069] 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 beta-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 beta-cyclodextrin can occur within the microbial host cell.

[0070] 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 (e.g., this variant cyclodextrin glucanotransferase can contain or consist of the amino acid sequence according to SEQ ID NO: 28), 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.

[0071] 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 beta-cyclodextrin tends to decrease. This is presumably due to the leakage of the enzyme 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 leakage. 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.

[0072] The inventors have found that enzyme stability can be improved by using freeze-dried enzymes, spray-drying the enzymes, and / or introducing additives.

[0073] 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.

[0074] In some embodiments, the cell slurry or whole cell lysate further comprises an additive. In some embodiments, the additive is selected from the group consisting of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, and combinations thereof. In some embodiments, the additive is added in an amount of about 0.1 w / v% to about 10 w / v% of the cell slurry or whole cell lysate, such as about 0.5 w / v% to about 5 w / v%. For example, the additive may be 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 additive is mannitol, sorbitol, sucrose, or a combination thereof.

[0075] 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 2 days. Methods of lyophilization are known in the art.

[0076] The inventors have found that adding an additive 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 additive, 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.

[0077] In some embodiments, the methods described herein produce a composition comprising at least 18 g / L of beta-cyclodextrin. In some embodiments, the method produces a composition comprising at least 25 g / L of beta-cyclodextrin, at least 30 g / L of beta-cyclodextrin, at least 40 g / L of beta-cyclodextrin, at least 50 g / L of beta-cyclodextrin, or at least 60 g / L of beta-cyclodextrin. In a preferred embodiment, the methods described herein produce a composition comprising at least 50 g / L of beta-cyclodextrin.

[0078] In some embodiments, the yield percentage of beta-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 yield percentage is calculated by dividing the total amount of beta-cyclodextrin produced by the methods described herein by the maximum theoretical amount of beta-cyclodextrin that could be produced from the sucrose reagent of the starting material.

[0079] Also provided herein is a composition comprising cyclodextrin, said cyclodextrin comprising beta-cyclodextrin and optionally further comprising alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof, and said composition comprising cyclodextrin comprises beta-cyclodextrin in an amount and / or concentration higher than alpha-cyclodextrin, gamma-cyclodextrin, or both. Preferably, the composition is obtained from the method provided herein. In some cases, the composition does not contain alpha-cyclodextrin and / or gamma-cyclodextrin. Preferably, the composition has a ratio of beta-cyclodextrin to alpha-cyclodextrin 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, a ratio of beta-cyclodextrin to gamma-cyclodextrin 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, or both a ratio of beta-cyclodextrin to gamma-cyclodextrin and a ratio of beta-cyclodextrin to alpha-cyclodextrin 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. Preferably, the composition has a ratio of beta-cyclodextrin to alpha-cyclodextrin of at least 10:1, such as 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, a ratio of beta-cyclodextrin to gamma-cyclodextrin of at least 10:1, such as 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, or both a ratio of beta-cyclodextrin to gamma-cyclodextrin and a ratio of beta-cyclodextrin to alpha-cyclodextrin of at least 10:1, such as 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.

[0080] 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 a higher amount and / or concentration of beta-cyclodextrin than a wild-type enzyme capable of converting amylose to cyclodextrin, the composition comprising cyclodextrin comprises beta-cyclodextrin and may further comprise alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof, the ratio of beta-cyclodextrin in the composition to alpha-cyclodextrin, gamma-cyclodextrin, or both is at least 10:1, 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 and an organic solvent (preferably toluene), and the entire reaction is carried out over a period of 8 hours or less.

[0081] Also provided herein is beta-cyclodextrin. Preferably, the beta-cyclodextrin is obtained from the method provided herein.

[0082] Also provided herein is the use of sucrose as a starting material for the production of beta-cyclodextrin. Also provided herein is the use of sucrose in a method for producing beta-cyclodextrin, the method not using starch.

[0083] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for converting sucrose to amylose, as described herein.

[0084] Also provided herein is the use of any one of the variant enzymes described herein for converting amylose to cyclodextrin and / or for producing beta-cyclodextrin in an amount and / or concentration higher than that of alpha-cyclodextrin, gamma-cyclodextrin, or both, as described herein.

[0085] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for the production of beta-cyclodextrin, which production does not require starch as a starting material.

[0086] 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: 1-48. 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: 1-48.

[0087] 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 48. 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 48.

[0088] 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.

[0089] 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.

[0090] Preferably, this enzyme is a variant cyclodextrin glucanotransferase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 28 - 30 or 35 - 47. 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: 28 - 30 or 35 - 47.

[0091] Also provided herein are enzyme compositions comprising one or more of the enzymes described herein.

[0092] 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.

[0093] Also provided herein is the use of an organic solvent, preferably toluene, to increase the yield of beta-cyclodextrin obtained in a method for producing beta-cyclodextrin, such as beta-cyclodextrin obtained from any one of the methods described herein.

[0094] Purification method Also provided herein is a method for purifying beta-cyclodextrin, the method comprising: i. preparing a crude composition containing beta-cyclodextrin; ii. obtaining a first precipitate containing beta-cyclodextrin from the crude composition, for example, by filtering the crude composition, subjecting the crude composition to centrifugation, subjecting the crude composition to a sedimentation operation, and / or washing with water; iii. dissolving the first precipitate, for example, by dissolving the first precipitate in water to obtain a first solution containing beta-cyclodextrin; iv. filtering the first solution to obtain a second solution containing beta-cyclodextrin; and v. crystallizing and / or precipitating the second solution to obtain a purified beta-cyclodextrin composition.

[0095] Step (ii) and / or (iv) The filtration step (iv) can remove insoluble substances.

[0096] In some embodiments, step (ii) and / or (iv) includes filtering and washing the substance obtained by filtration with, for example, water or alkaline water.

[0097] In some embodiments, step (iv) includes filtration with a filter aid. In some embodiments, the filter aid includes silicon dioxide. An example of a suitable filter aid is 1% Celite® commercially available from Sigma-Aldrich. The use of a filter aid can be advantageous for shortening the overall filtration time of step (iv).

[0098] The filtration step (iv) can be carried out at a temperature of about 4°C to about 25°C.

[0099] Dissolution step (iii) In some embodiments, step (iii) includes dissolving the first precipitate in an alkaline solution. The precipitate may be dissolved in NaOH, such as 1M NaOH, for example, by adding a plurality (e.g., 5) volumes of 1M NaOH.

[0100] In some embodiments, step (iii) may include heating the solution until beta-cyclodextrin dissolves. For example, this may require heating the solution to about 60°C or higher, such as about 65°C or higher, such as about 70°C or higher, such as about 75°C or higher. Next, the temperature of the solution may be lowered, for example, by about 5°C or more, before subsequent steps.

[0101] Crystallization step (v) Step (v) may include neutralizing the second solution. Optionally, the neutralization includes the addition of HCl. For example, the neutralization may include the addition of 6M HCl.

[0102] Step (v) may include seeding the second solution with crystalline beta-cyclodextrin.

[0103] In some embodiments, step (v) may further include heating the solution until the beta-cyclodextrin dissolves. For example, this may require heating the solution to about 60 °C or higher, such as about 65 °C or higher, such as about 70 °C or higher, such as about 75 °C or higher. In a preferred embodiment, the solution is heated to about 75 °C. Next, before adding the crystalline beta-cyclodextrin as seed crystals, the temperature of the solution may be decreased, for example, decreased by about 5 °C or more. In a preferred embodiment, the solution is heated to about 75 °C and then decreased to about 70 °C before adding the seed crystals.

[0104] In some embodiments, step (v) may include cooling the solution to below room temperature, such as about 20 °C or lower, about 15 °C or lower, about 10 °C or lower, or about 5 °C or lower, after adding the seed crystals. In a preferred embodiment, the solution is cooled to about 4 °C. In some embodiments, the solution is cooled over about 1 to about 12 hours. In some embodiments, the solution is cooled over about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In a preferred embodiment, the solution is cooled to about 4 °C over about 4 hours.

[0105] The solution after adding the seed crystals may be maintained under conditions suitable for beta-cyclodextrin crystal formation. For example, the solution may be maintained at below room temperature, such as about 20 °C or lower, about 15 °C or lower, about 10 °C or lower, or about 5 °C or lower. In a preferred embodiment, the solution is maintained at about 4 °C. In some embodiments, the solution is maintained at below room temperature for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, or at least about 12 hours. Preferably, the solution is maintained at about 4 °C for 12 hours or more.

[0106] The crystallization step (v) may include a filtration step. The filtration step may include vacuum filtration.

[0107] In some embodiments, step (v) further comprises washing the composition with water.

[0108] In some embodiments, step (v) further comprises drying the composition, optionally drying the composition at about 45 °C (e.g., in a vacuum oven).

[0109] Precipitation step (v) Step (v) may include neutralizing the second solution, optionally the neutralization includes the addition of HCl. For example, the neutralization may include the addition of about 6 M HCl.

[0110] Step (v) may include the addition of an antisolvent. The antisolvent may increase the yield of purified beta-cyclodextrin in the composition obtained by the purification method. The antisolvent is a solvent in which beta-cyclodextrin is poorly soluble, for example, a solvent in which beta-cyclodextrin does not dissolve at about 50 °C and about 60 °C. The antisolvent may be THF, AcN, EtOH, toluene, acetone, or a mixture of acetone and water (e.g., a 10:90, or 20:80, or 30:70, or 40:60, or 50:50, or 60:40, or 70:30, or 80:20, or 90:10 acetone:water mixture). In some embodiments, when the antisolvent is a mixture of acetone and water, this mixture may be 10-90%, 20-80%, 30-70%, 40-60%, or about 50% acetone. Preferably, the antisolvent used is a mixture of acetone and water, for example, a 50% acetone and 50% water mixture.

[0111] In some embodiments, step (v) may include cooling the solution to a temperature below room temperature, such as below about 20 °C, below about 15 °C, below about 10 °C, or below about 5 °C, after the addition of the antisolvent. In a preferred embodiment, the solution is cooled to about 4 °C. In some embodiments, the solution is cooled over a period of about 1 to about 12 hours. In some embodiments, the solution is cooled for about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In a preferred embodiment, the solution is cooled to about 4 °C over a period of about 4 hours.

[0112] The solution may be maintained under conditions suitable for β-cyclodextrin precipitate formation. For example, the solution may be maintained at a temperature below room temperature, such as below about 20 °C, below about 15 °C, below about 10 °C, or about 5 °C. In a preferred embodiment, the solution is maintained at about 4 °C. In some embodiments, the solution is maintained below room temperature for at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 6 hours, at least about 7 hours, at least about 8 hours, at least about 9 hours, at least about 10 hours, at least about 11 hours, or at least about 12 hours. Preferably, the solution is maintained at about 4 °C for 12 hours or more.

[0113] In some embodiments, the solution is cooled to about 4 °C over a period of about 4 hours and then maintained at about 4 °C for about 12 hours.

[0114] The precipitation in step (v) may include a filtration step. The filtration step may include vacuum filtration.

[0115] In some embodiments, step (v) further includes washing the composition with water.

[0116] In some embodiments, step (v) further includes drying the composition, optionally drying the composition at about 45 °C (e.g., under vacuum).

[0117] Composition Preferably, the crude composition of step (i) is obtained by any one of the enzymatic methods described and claimed herein.

[0118] In some embodiments, the crude composition is cooled prior to step (ii). For example, the crude composition may be cooled to room temperature for at least about 3 hours and then to about 4 °C for at least about 3 hours.

[0119] Also provided herein is a purified beta-cyclodextrin composition. The purified beta-cyclodextrin composition can be obtained from any one of the purification methods described and claimed herein. The beta-cyclodextrin in the composition can have a purity of 75 wt% or more, such as 80 wt% or more, such as 85 wt% or more, such as 90 wt% or more, or such as 95 wt% or more.

[0120] The purity of beta-cyclodextrin is 1 measurable by 1H-NMR and can provide the anhydrous amount of beta-cyclodextrin.

[0121] Preferably, the purified beta-cyclodextrin composition consists essentially of beta-cyclodextrin and optional water, preferably consists of beta-cyclodextrin and optional water, and preferably consists of beta-cyclodextrin. The purified beta-cyclodextrin composition may contain 2 wt% or less of toluene, for example, may not contain toluene. The purified beta-cyclodextrin composition may contain 1 wt% or less of sucrose, fructose and / or amylose, for example, may not contain sucrose, fructose and / or amylose. The purified beta-cyclodextrin composition may contain 5 wt% or less, preferably 1 wt% or less, preferably alpha and / or gamma-cyclodextrin, for example, may not contain alpha and / or gamma-cyclodextrin.

[0122] The recovery rate of beta-cyclodextrin by the purification method described in this specification can be at least 50%, at least 60%, at least 70%, or at least 80%. In other words, the amount of beta-cyclodextrin in the purified composition can be at least 50% (or at least 60%, at least 70%, or at least 80%) of the amount of beta-cyclodextrin in the crude composition. The amounts of beta-cyclodextrin and any other components in the composition can be 1 measured by 1H-NMR (in weight %) or by HLPC-ELSD (in g / L).

[0123] Glossary 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 (polynucleotide or amino acid) 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 advanced 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 percent identity over the full length of the proteins being compared. The default parameters are set, for example, to optimize searches with short query sequences using 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 therebetween. Generally, the present disclosure encompasses sequences having at least about 70%, 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.

[0124] 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 of 8.5 to 11.5.

[0125] 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".

[0126] As used herein, "a", "an", and "the" can include plural referents unless explicitly and specifically limited to one referent.

[0127] The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0128] 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.

[0129] 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 beta-cyclodextrin than the wild-type enzyme capable of converting amylose to cyclodextrin, the composition containing cyclodextrin contains beta-cyclodextrin and may further contain, optionally, alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof, and the composition containing cyclodextrin contains a higher amount and / or concentration of beta-cyclodextrin than alpha-cyclodextrin, gamma-cyclodextrin, or both.

[0130] Embodiment 2: The method according to Embodiment 1, wherein the enzyme in (a) is amylosucrase or the enzyme mixture in (a) contains amylosucrase.

[0131] 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.

[0132] 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 than wild-type amylosucrase.

[0133] Embodiment 5: The method according to Embodiment 3 or 4, wherein the wild-type amylosucrase is Cellulomonas carboniz T26 amylosucrase.

[0134] Embodiment 6: The method according to Embodiment 5, wherein the wild-type amylosucrase comprises the amino acid sequence of SEQ ID NO: 1.

[0135] Embodiment 7: The method according to Embodiment 3 or 4, wherein the wild-type amylosucrase is Neisseria polysaccharea amylosucrase.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] 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.

[0140] 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.

[0141] Embodiment 13: The method according to Embodiment 1, wherein the enzyme mixture in (a) comprises at least two enzymes capable of converting sucrose into amylose in combination or collectively.

[0142] Embodiment 14: The method according to Embodiment 13, wherein the enzyme mixture comprises sucrose phosphorylase.

[0143] Embodiment 15: The method according to Embodiment 14, wherein the sucrose phosphorylase is capable of converting sucrose into glucose-1-phosphate.

[0144] 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 into glucose-1-phosphate.

[0145] 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.

[0146] Embodiment 18: The method according to any one of Embodiments 14 to 17, 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.

[0147] Embodiment 19: The method according to any one of Embodiments 13 to 18, wherein the enzyme mixture comprises alpha-glucan phosphorylase.

[0148] Embodiment 20: The method according to embodiment 19, wherein the alpha - glucan phosphorylase can convert the glucose - 1 - phosphate into amylose.

[0149] 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 to amylose.

[0150] 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.

[0151] 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 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.

[0152] 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.

[0153] 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.

[0154] Embodiment 26: The method according to embodiment 25, wherein the wild-type cyclodextrin glucanotransferase is Bacillus sp. strain number 38-2 cyclodextrin glucanotransferase.

[0155] Embodiment 27: The method according to embodiment 26, wherein the Bacillus sp. strain number 38-2 cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of SEQ ID NO: 25.

[0156] Embodiment 28: The method according to embodiment 25, wherein the wild-type cyclodextrin glucanotransferase is Bacillus circulans strain 251 cyclodextrin glucanotransferase.

[0157] Embodiment 29: The method according to embodiment 28, wherein the Bacillus circulans strain 251 cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of SEQ ID NO: 26 or 27, for example, the amino acid sequence of SEQ ID NO: 27.

[0158] Embodiment 30: The method according to any one of embodiments 24 to 29, wherein the variant cyclodextrin glucanotransferase comprises or consists 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: 25 to 27.

[0159] Embodiment 31: The method according to any one of embodiments 28 to 30, wherein the at least one amino acid variant comprises at least one amino acid substitution with respect to the wild-type cyclodextrin glucanotransferase.

[0160] Embodiment 32: The method according to embodiment 31, wherein the at least one amino acid substitution comprises an amino acid substitution at amino acid position 31 with respect to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 27.

[0161] Embodiment 33: The method according to embodiment 32, wherein the amino acid substitution at position 31 of the amino acid is selected from the group consisting of A31R, A31P, and A31T.

[0162] Embodiment 34: The method according to any one of embodiments 1 to 33, wherein the contacting in (a) and the contacting in (b) are performed sequentially.

[0163] Embodiment 35: The method according to any one of embodiments 1 to 33, wherein the contacting in (a) and the contacting in (b) are performed simultaneously or substantially simultaneously.

[0164] Embodiment 36: The method according to any one of embodiments 1 to 35, wherein the amylose produced in (a) is not purified or isolated before the contacting in (b).

[0165] Embodiment 37: The method according to any one of embodiments 1 to 36, wherein the contacting in (a), the contacting in (b), or both are performed in vitro.

[0166] Embodiment 38: The method according to embodiment 37, wherein the contacting in (a), the contacting in (b), or both are performed in a container, vial, bottle, test tube, well, plate, or wrapper.

[0167] Embodiment 39: The method according to embodiment 37 or 38, 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.

[0168] Embodiment 40: The method according to any one of embodiments 37 to 39, 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.

[0169] Embodiment 41: The method according to any one of Embodiments 1 to 40, wherein the contacting in (a), the contacting in (b), or both are carried out in vivo.

[0170] Embodiment 42: The method according to Embodiment 41, wherein the contacting in (a), the contacting in (b), or both are carried out in a recombinant host cell.

[0171] Embodiment 43: The method according to Embodiment 42, 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.

[0172] Embodiment 44: The method according to Embodiment 42 or 43, wherein the recombinant host cell is a microbial cell.

[0173] Embodiment 45: The method according to Embodiment 44, wherein the microbial cell is a bacterial cell.

[0174] Embodiment 46: The method according to any one of Embodiments 1 to 45, wherein the ratio of beta-cyclodextrin to alpha-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

[0175] Embodiment 47: The method according to any one of Embodiments 1 to 46, wherein the ratio of beta-cyclodextrin to gamma-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

Examples

[0176] Example 1. One - pot synthesis of beta - cyclodextrin from sucrose using wild - type amylosucrase and variant cyclodextrin glucanotransferase enzymes In this example, amylosucrase (having the amino acid sequence according to SEQ ID NO: 2) and variant cyclodextrin glucanotransferase enzyme (having the amino acid sequence according to SEQ ID NO: 28) were expressed in Escherichia coli and then separated from the insoluble cell debris mixture by centrifugation. The two enzymes were exposed to sucrose at various concentrations (150 g / L, 200 g / L, and 250 g / L). In this example, both enzymes were simultaneously exposed to the substrate sucrose. The enzymes were added only once. All reactions were carried out at 45 °C in 0.1 M sodium citrate buffer. The amounts of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin were measured by HPLC. Figure 4 shows that the one-pot synthesis reaction (including amylosucrase and variant cyclodextrin glucanotransferase enzyme) can produce beta-cyclodextrin from sucrose at concentrations above 18 g / L.

[0177] Example 2. One - pot synthesis of beta - cyclodextrin from sucrose using variant amylosucrase and variant cyclodextrin glucanotransferase enzymes In this example, different variant amylosucrase enzymes were combined with a variant cyclodextrin glucanotransferase enzyme (having the amino acid sequence according to SEQ ID NO: 28) and exposed to sucrose. More specifically, the different variant amylosucrase enzymes used were "R234Q" (having the amino acid sequence according to SEQ ID NO: 3), "R234G" (having the amino acid sequence according to SEQ ID NO: 4), "R234NΔ8AA" (having the amino acid sequence according to SEQ ID NO: 48), "R234A" (having the amino acid sequence according to SEQ ID NO: 5), "R234C" (having the amino acid sequence according to SEQ ID NO: 8), and "R234" (having the amino acid sequence according to SEQ ID NO: 2). Both enzymes were simultaneously exposed to the substrate sucrose. In this example, the enzymes were added only once. All reactions were carried out at 45 °C in 0.1 M sodium citrate buffer. The ratio of the amylosucrase enzyme to the cyclodextrin glucanotransferase enzyme was 20:1 in all examples. The amounts of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin were measured by HPLC. Figure 5 shows that a one-pot synthesis reaction (including variant amylosucrase and variant cyclodextrin glucanotransferase enzymes) can produce beta-cyclodextrin from sucrose at a concentration exceeding 18 g / L. Example 3. One - pot synthesis of beta - cyclodextrin from sucrose using a three - enzyme system In this example, beta-cyclodextrin was produced from sucrose using a three-enzyme system (i.e., method step (a) was a two-enzyme method and method step (b) was a one-enzyme method). Each enzyme of sucrose phosphorylase (having the amino acid sequence according to SEQ ID NO: 20), alpha-glucan phosphorylase (having the amino acid sequence according to SEQ ID NO: 24), and cyclodextrin glucanotransferase (having the amino acid sequence according to SEQ ID NO: 28) was expressed in Escherichia coli and then separated from the insoluble cell debris mixture by centrifugation. In this example, all the enzymes were simultaneously exposed to the substrate sucrose. The enzymes were added only once. In this example, the amounts of sucrose phosphorylase, alpha-glucan phosphorylase, and sucrose were kept constant, and the amount of cyclodextrin glucanotransferase was varied in the same manner as the reaction time. FIG. 6 shows that a one-pot synthesis reaction using three enzymes (sucrose phosphorylase having the amino acid sequence according to SEQ ID NO: 20, alpha-glucan phosphorylase having the amino acid sequence according to SEQ ID NO: 24, and cyclodextrin glucanotransferase having the amino acid sequence according to SEQ ID NO: 28) can produce beta-cyclodextrin from sucrose at a concentration exceeding 1 g / L. All reactions were carried out at 50° C. in 0.1 M Tris-HCl at pH 7.4.

[0178] Example 4. Purification of beta - cyclodextrin In this example, as described herein, a crude composition containing beta-cyclodextrin was produced by a two-enzyme system comprising each enzyme of amylosucrase (having the amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having the amino acid sequence according to SEQ ID NO: 28). This crude composition was purified by the following method.

[0179] The total productivity titer of the crude composition was 64.2 g / L. The first precipitate was obtained by filtration. Next, the first precipitate was dissolved by adding 200 mL of 1M NaOH to obtain a first solution. The first solution was filtered to remove insoluble substances and then neutralized with approximately 30 mL of 6M HCl to obtain a second solution. The second solution was heated to 60 °C to dissolve beta-cyclodextrin and then the temperature was lowered to 55 °C. Seeding of the solution was carried out by adding 0.5 g of crystalline beta-cyclodextrin to the solution. After 30 minutes, the temperature was lowered to 4 °C over 4 hours and held overnight for about 12 hours. A purified beta-cyclodextrin composition was obtained.

[0180] As shown in Figure 7, this composition was 1 evaluated by 1H-NMR. This composition consisted of only beta-cyclodextrin and water. Beta-cyclodextrin had a purity of 90.5% as measured by 1H-NMR analysis. 1 No other impurities (except for the presence of water) were identified by 1H-NMR or HPLC-ELSD. 1 The recovery rate of beta-cyclodextrin from this purification method was 80.1% (i.e., the amount of purified beta-cyclodextrin in the final composition was 80.1% of the amount of beta-cyclodextrin in the crude composition). These amounts were measured by HPLC from the dry mass of the composition.

[0181] This example demonstrates that the purification method of the present invention provides a purified beta-cyclodextrin composition in which beta-cyclodextrin advantageously has a high purity. The recovery rate of beta-cyclodextrin is also improved.

[0182]

[0183] Example 5. One - pot synthesis of beta - cyclodextrin from sucrose using a two - enzyme system in a 10L STR ​In this example, in a 5 L reaction, a two-enzyme system containing each of amylosucrase (having the amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having the amino acid sequence according to SEQ ID NO: 28) was used to produce beta-cyclodextrin from sucrose.

[0184] More specifically, 266 g of Na2HPO4 and 20.1 g of citric acid were added to a 10 L stirred tank reactor (STR) containing 4.65 L of H2O. The solution was stirred until completely dissolved. A total of 1.25 kg of sucrose was added to the solution, which was then heated to 45 °C and the pH was adjusted to 7.0 by adding 6 M HCl. Once this temperature was reached, 330 mL of a whole cell lysate containing 10.4 U / mL of amylosucrase was added, followed by 17 mL of a whole cell lysate containing cyclodextrin glucanotransferase. After 1 hour, 400 mL of toluene was added. The reaction mixture was stirred at 160 rpm for an additional 6 hours, after which the reactor was cooled to 4 °C and held overnight for approximately 12 hours.

[0185] The precipitate was collected by vacuum filtration through a sintered funnel and washed with approximately 150 mL of H2O. The solid was then partially dried in vacuo to obtain 678 g of a wet crude material (containing 52 wt% β-CD and 6 wt% toluene ([ 1 measured by 1H-NMR), a cake volume of about 990 cm 3 ). The titer of the final reaction was 67.9 g / L of beta-cyclodextrin.

[0186] This example demonstrates that a scaled-up one-pot synthesis of beta-cyclodextrin from sucrose using a two-enzyme system produces beta-cyclodextrin in high yield.

[0187] Example 6. Purification of beta - cyclodextrin from crude precipitate In this example, the crude composition produced by the enzymatic method of Example 5 was purified. Example 5 describes preparing a crude composition containing beta-cyclodextrin and obtaining a first precipitate by vacuum filtration and washing with water.

[0188] The first precipitate was then dissolved in 1.7 L of 1 M NaOH to obtain a first solution, and the first solution was then filtered through a pad of Celite® to obtain a second solution. The second solution was neutralized with approximately 400 mL of 6 M HCl. Next, the second solution was heated to 75° C. until dissolved, and then the temperature was lowered to 70° C. Next, seeding was performed on the second solution by adding 3 g of crystalline β-CD, and after leaving it standing for 30 minutes, the solution was cooled to 4° C. over 4 hours. The solution was held at 4° C. overnight for about 12 hours. Next, the composition was collected under vacuum and washed with H2O. Next, the composition was dried in a vacuum oven at 45° C. As shown in FIGS. 8 and 9, HPLC-ELSD and 1 1H-NMR were used to evaluate the purity.

[0189] This composition consisted of beta-cyclodextrin, water, and a trace amount (0.23 wt %) of toluene. No other components were observed by HPLC-ELSD or 1 1H-NMR. Beta-cyclodextrin had a purity of 91.7 wt % as measured by 1 1H-NMR. No other impurities were identified by HPLC-ELSD or 1 1H-NMR. 302 g of beta-cyclodextrin was obtained. Thus, the recovery rate of beta-cyclodextrin was 78%.

[0190] This example demonstrates that the purification method of the present invention results in a purified beta-cyclodextrin composition with a high recovery rate of beta-cyclodextrin.

[0191] Example 7. Purification of beta - cyclodextrin using acetone as an antisolvent 1 15 g of a crude composition containing beta-cyclodextrin with a purity of 87% as measured by 1H-NMR was prepared. A first precipitate was obtained by filtering the crude composition.

[0192] The first precipitate was dissolved in 75 mL of 1 M NaOH to obtain a first solution, which was then filtered to remove insoluble substances. A mixture of 10% acetone and 90% water was added, and then the second solution was neutralized with 6 M HCl and cooled to 4 °C over about 2 hours. The second solution was filtered by vacuum filtration, washed with water, and then washed with acetone. This substance was dried, 1 9.0 g of beta-cyclodextrin with a purity of 93.7 wt% was obtained by measurement using H-NMR. Therefore, the recovery rate of beta-cyclodextrin was 64.6%.

[0193] The above purification process was repeated except that a mixture of 50% acetone and 50% water was added instead. This substance was dried, 1 10.72 g of beta-cyclodextrin with a purity of 98.8 wt% was obtained by measurement using H-NMR. Therefore, the recovery rate of beta-cyclodextrin was 81.2%.

[0194] These compositions were 1 Evaluated by H-NMR. The results are shown in the following table, and the spectra are shown in Figure 10. By NMR analysis, it was confirmed that only beta-cyclodextrin, water, toluene, and acetone were present in the composition containing the purified beta-cyclodextrin.

Table 6

[0195] Example 8. Freeze - drying of cell slurries and cell lysates of amylosucrase and cyclodextrin glucanotransferase containing additives The lysates and whole cell slurries of amylosucrase (having the amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (having the amino acid sequence according to SEQ ID NO: 28) were lyophilized together with various additives. More specifically, 0.5 w / v%, 1 w / v%, or 5 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. Next, the mixture was lyophilized over a period of 2 days.

[0196] 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.

[0197] 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 concentration of starch was quantified by spectrophotometric analysis. 1 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 activity was compared with that of the non-lyophilized cell lysate (or the whole cell slurry respectively). The results are shown in FIGS. 11A and 11B respectively.

[0198] Enzymatic activity of cyclodextrin glucanotransferase To 2.998 mL of a solution of soluble starch (10 g / L) dissolved in a sodium citrate buffer (0.1 M) at pH 7 and 45°C, 2 μL of a cyclodextrin glucanotransferase cell-free lysate was added. The solution was shaken at 1200 rpm for 1 hour. 1 activity unit (U / mL) was defined as the amount of enzyme required to produce 1 mmol of beta-cyclodextrin per minute at pH 7 and 45°C. The activity was compared with that of the non-lyophilized cooled cell-free lysate. The results are shown in FIG. 12.

[0199] As shown in FIGS. 11 to 12, when additives are added to amylosucrase and cyclodextrin glucanotransferase before lyophilization, the stability of the enzymes is improved. In particular, the addition of 5% sucrose, 0.5% mannitol, and 0.5% sorbitol has been demonstrated to improve the enzyme activity retention rate of cell lysates and whole cell slurries of amylosucrase and cyclodextrin glucanotransferase.

[0200] Example 9. Influence of organic solvents on the synthesis of beta - cyclodextrin from sucrose In this example, a variant amylosucrase enzyme (having the amino acid sequence according to SEQ ID NO: 3) was combined with a variant cyclodextrin glucanotransferase enzyme (having the amino acid sequence according to SEQ ID NO: 28) and exposed to sucrose. Both enzymes were simultaneously exposed to the substrate sucrose. In this example, the enzymes were added only once. The reaction volume was 500 mL and was carried out under the standard conditions described herein. In the absence of an organic solvent, beta-cyclodextrin was obtained at a concentration of about 18 g / L.

[0201] Next, 40 mL, 80 mL, or 160 mL of toluene was added to the reaction mixture, respectively, to perform different examples. In each of these examples, the addition of toluene resulted in a significant increase in the yield of beta-cyclodextrin. Specifically, when the enzyme reaction was carried out in a reaction mixture containing toluene, beta-cyclodextrin was obtained at a concentration of about 70 g / L.

[0202] The foregoing 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. This example, in conjunction with the methods described herein, represents the preferred embodiments at the present time, is exemplary, and is 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.

[0203] 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 included.

Claims

1. A method for producing a composition containing cyclodextrin, wherein the method is (a) Contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that enable 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 enable the conversion of amylose to cyclodextrin, thereby producing the composition containing cyclodextrin. Includes, The enzyme in (b) that can convert amylose to cyclodextrin is a variant enzyme that can produce beta-cyclodextrin in a higher amount and / or concentration than alpha-cyclodextrin, gamma-cyclodextrin, or both, compared to the wild-type enzyme that can convert amylose to cyclodextrin. The composition comprising cyclodextrin comprises beta-cyclodextrin and may optionally further comprise alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof. The method wherein the composition comprising cyclodextrin comprises beta-cyclodextrin in an amount and / or concentration higher than that of alpha-cyclodextrin, gamma-cyclodextrin, or both.

2. (a) The enzyme is amylosculase, or the enzyme mixture in (a) contains amylosculase. Optionally, 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 compared to wild-type amylosucrase. Furthermore, optionally, A) The wild-type amylosculase described above (i) Cellulomonas carboniz T26 amylosculase, for example, the wild-type amylosculase containing the amino acid sequence of SEQ ID NO: 1, or (ii) Neisseria polysaccharea amylosculase, for example, the wild-type amylosculase contains the amino acid sequence of SEQ ID NO: 2 B) The variant amylosculase contains 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, and / or C) The at least one amino acid variant comprises at least one amino acid substitution relative to wild-type amylosculase, Preferably, the at least one amino acid substitution includes an amino acid substitution at the 234 position relative to the wild-type amylosculase 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 method according to claim 1, wherein the amino acid substitution at position 234 is R234Q.

3. (a) The enzyme mixture comprises at least two enzymes that can convert sucrose to amylose in combination or collectively, Optionally, the enzyme mixture may include sucrose phosphorylase. Preferably, the sucrose phosphorylase can convert sucrose to glucose-1-phosphate. Preferably, (a) further comprises contacting the sucrose with the sucrose phosphorylase under conditions that enable the conversion of the sucrose to glucose-1-phosphate, Furthermore, optionally, (i) The sucrose phosphorylase is selected from the group consisting of Bifidobacterium longum sucrose phosphorylase, Leuconostococcus mesenteroides sucrose phosphorylase, and Streptococcus mutans sucrose phosphorylase, and / or (ii) The method according to claim 1, wherein the sucrose phosphorylase comprises or consists 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: 17 to 20, or any one of the amino acid sequences of SEQ ID NOs: 17 to 20.

4. The enzyme mixture comprises alpha-glucan phosphorylase, Preferably, the alpha-glucan phosphorylase can convert the glucose-1-phosphate to amylose. Preferably, (a) further comprises contacting the glucose-1-phosphate with the alpha-glucan phosphorylase under conditions that enable the conversion of the glucose-1-phosphate to amylose, Optionally, A) 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, and / or B) The method according to claim 3, wherein the alpha-glucan phosphorylase comprises or consists 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. 21 to 24.

5. The method according to any one of claims 1 to 4, wherein the enzyme capable of converting the amylose in (b) to cyclodextrin comprises a variant cyclodextrin glucanotransferase.

6. The variant cyclodextrin lucanotransferase comprises at least one amino acid variant relative to the wild-type cyclodextrin lucanotransferase, Selectively, the wild-type cyclodextrin glucanotransferase is used. (i) Bacillus sp. strain number 38-2 is a cyclodextrin glucanotransferase, Preferably, the Bacillus sp. strain 38-2 cyclodextrin glucanotransferase contains or consists of the amino acid sequence of SEQ ID NO: 25, or (ii) Bacillus circulans strain 251 cyclodextrin glucanotransferase, Preferably, the method according to claim 5, wherein the Bacillus circulans strain 251 cyclodextrin glucanotransferase contains or comprises the amino acid sequence of SEQ ID NO: 26 or 27, for example, SEQ ID NO:

27.

7. The method according to claim 5, wherein the variant cyclodextrin glucanotransferase comprises or consists 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. 25 to 27.

8. The variant cyclodextrin lucanotransferase comprises at least one amino acid variant relative to wild-type cyclodextrin lucanotransferase, The at least one amino acid variant comprises at least one amino acid substitution relative to the wild-type cyclodextrin glucanotransferase, The wild-type cyclodextrin glucanotransferase is Bacillus circulans strain 251 cyclodextrin glucanotransferase, Preferably, the Bacillus circulans strain 251 cyclodextrin glucanotransferase contains or comprises the amino acid sequence of SEQ ID NO: 26 or 27, for example, SEQ ID NO:

27. Preferably, the at least one amino acid substitution includes an amino acid substitution at the 31st amino acid position relative to the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO:

27. Preferably, the amino acid substitution at the 31st position of the amino acid is selected from the group consisting of A31R, A31P, and A31T, according to claim 5.

9. A) The contact in (a) and the contact in (b) are performed either (i) sequentially or (ii) simultaneously or substantially simultaneously. B) The amylose produced in (a) is not purified or isolated before contact in (b), and / or C) The contact in (a), the contact in (b), or both are performed in vitro. Optionally, (I) The contact in (a), the contact in (b), or both is performed in a container, vial, bottle, test tube, well, plate, or packaging, and / or (II) At least one of the enzymes or enzyme mixtures in (a), the variant enzyme in (b), or both, is a purified enzyme, an isolated enzyme, or both. Preferably, the method according to any one of claims 1 to 4, wherein at least one enzyme from the enzyme or the enzyme mixture of (a), the variant enzyme of (b), or both are recombinantly produced enzymes.

10. (a) the contact, (b) the contact, or both are performed in vivo. Selectively, (a) the contact, (b) the contact, or both are performed in recombinant host cells. Preferably, the recombinant host cell comprises heterologous nucleic acids encoding at least one of the enzymes in (a) or the enzyme mixture, the variant enzyme in (b), or both. Preferably, the recombinant host cell is a microbial cell. Preferably, the method according to any one of claims 1 to 4, wherein the microbial cell is a bacterial cell.

11. The ratio of beta-cyclodextrin to alpha-cyclodextrin in the composition containing cyclodextrin is at least 2:1, preferably at least 100:1, and / or The method according to any one of claims 1 to 4, wherein the ratio of beta-cyclodextrin to gamma-cyclodextrin in the composition containing cyclodextrin is at least 2:1, preferably at least 100:

1.

12. A) At least one enzyme from the enzyme or the enzyme mixture of (a), the variant enzyme of (b), or both are produced in Pichia yeast cells. B) The contact in (a) and / or the contact in (b) is carried out in a reaction mixture containing an organic solvent, preferably toluene. C) At least one of the enzymes or enzyme mixtures in (a), the variant enzyme in (b), or both are immobilized on the resin, and / or D) At least one of the enzymes or enzyme mixtures of (a), the variant enzyme of (b), or both are provided in a cell slurry or a whole cell lysate. Optionally, the cell slurry or the whole cell lysate further contains an additive selected from the group consisting of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, and combinations thereof. Preferably, the additive is selected from the group consisting of mannitol, sorbitol, sucrose, and / or combinations thereof. E) The method according to any one of claims 1 to 4, wherein the ratio of beta-cyclodextrin in the composition to alpha-cyclodextrin, gamma-cyclodextrin, or both is at least 10:1, 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 and toluene, and the entire reaction is carried out over a period of 8 hours or less.

13. A composition comprising a cyclodextrin, wherein the cyclodextrin comprises beta-cyclodextrin, and may optionally further comprise alpha-cyclodextrin, gamma-cyclodextrin, or any combination thereof, wherein the composition comprising the cyclodextrin comprises beta-cyclodextrin in an amount and / or concentration higher than that of alpha-cyclodextrin, gamma-cyclodextrin, or both, and is obtained from the method according to any one of claims 1 to 4.

14. The ratio of beta-cyclodextrin to alpha-cyclodextrin in the composition containing cyclodextrin is at least 2:1, preferably at least 100:

1. The composition according to claim 13, and / or the ratio of beta-cyclodextrin to gamma-cyclodextrin in the composition comprising cyclodextrin is at least 2:1, preferably at least 100:

1.

15. The composition according to claim 13, wherein the yield percentage of beta-cyclodextrin is at least about 10%, preferably at least about 60%.

16. The use of sucrose for the production of beta-cyclodextrin, wherein the method of production is the method according to any one of claims 1 to 4.

17. The use of one or more enzymes from SEQ ID NO: 1 to SEQ ID NO: 48 for the production of beta-cyclodextrin, wherein the method of production is the method according to any one of claims 1 to 4.

18. An enzyme containing or comprising one amino acid sequence from sequence numbers 1 to 48, or an enzyme containing or comprising an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with one amino acid sequence from sequence numbers 1 to 48.

19. (i) Variant amyloscase enzymes containing or consisting of one amino acid sequence from sequence numbers 3-16 or 48, (ii) Variant sucrose phosphorylase enzyme containing or consisting of the amino acid sequence of Sequence ID No. 20, (iii) A variant alpha-glucan phosphorylase enzyme containing or consisting of the amino acid sequence of Sequence ID No. 24, (iv) Variant cyclodextrin glucanotransferase enzyme containing or consisting of one amino acid sequence from sequence numbers 28-30 or 35-47 The enzyme according to claim 18.

20. A method for purifying beta-cyclodextrin, i. A step of preparing a crude composition containing beta-cyclodextrin, ii. A step of obtaining a first precipitate containing beta-cyclodextrin from the crude composition, iii. The step of dissolving the first precipitate to obtain a first solution containing beta-cyclodextrin, iv. The first solution is filtered to obtain a second solution containing beta-cyclodextrin. v. The second solution is crystallized and / or precipitated to obtain a purified beta-cyclodextrin composition. The method, including the method described above.

21. The method according to claim 20, wherein the crude composition of step (i) is a composition obtained by the method according to any one of claims 1 to 4.

22. A purified beta-cyclodextrin composition, wherein the purified beta-cyclodextrin composition is obtained by the method described in claim 20.

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