Process for producing gamma-cyclodextrin

Abstract

Provided herein is a method for the enzymatic production of gamma-cyclodextrin from sucrose. Optionally, the method comprises contacting sucrose with one or more enzymes to convert sucrose to amylose, and subsequently contacting the amylose with one or more enzymes to convert the amylose to gamma-cyclodextrin. Optionally, the method produces gamma-cyclodextrin in a high yield compared to alpha-cyclodextrin, beta-cyclodextrin, or both.

Description

Technical Field

[0001] [Background Art] Cyclodextrin is a kind of cyclic oligosaccharide composed of cyclic oligomers of glucose. Cyclodextrin has a lipophilic core and a hydrophilic outer surface, and thus is useful in the pharmaceutical industry and various other industries. Natural cyclodextrins (i.e., alpha-cyclodextrin, beta-cyclodextrin, and gamma-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 conversion of starch. However, standard production methods have various disadvantages, including supply chain shortages, scalability, quality variability, purification, and the cost of goods. Therefore, improved methods for producing cyclodextrin to address these problems are needed.

Summary of the Invention

[0002] There is an unmet need for methods to produce cyclodextrin without the conversion of starch as a starting material. The present disclosure meets this unmet need by providing a method for biosynthetically producing cyclodextrin without using starch as a starting material. 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 by-product waste, as well as by-products that may be useful for other purposes.

[0003] In one aspect, a method for producing a composition comprising cyclodextrin is provided, the method comprising: (a) contacting sucrose with an enzyme or enzyme mixture capable of converting sucrose to amylose under conditions that allow the conversion of sucrose to amylose, thereby producing amylose; (b) contacting the amylose produced in (a) with an enzyme capable of converting amylose to cyclodextrin under conditions that allow the conversion of amylose to cyclodextrin, thereby producing a composition comprising cyclodextrin, the composition comprising cyclodextrin comprising gamma-cyclodextrin and optionally further comprising alpha-cyclodextrin, beta-cyclodextrin, or any combination thereof, the composition comprising cyclodextrin comprising gamma-cyclodextrin in an amount and / or concentration higher than alpha-cyclodextrin, beta-cyclodextrin, or both. Optionally, the enzyme in (a) is amylosucrase or the enzyme mixture in (a) comprises amylosucrase. Optionally, the amylosucrase is a variant amylosucrase comprising at least one amino acid variant relative to wild-type amylosucrase. Optionally, the variant amylosucrase is capable of producing a higher amount and / or concentration of amylose from sucrose relative to wild-type amylosucrase. Optionally, the wild-type amylosucrase is Cellulomonas carboniz T26 amylosucrase. Optionally, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 1. Optionally, the wild-type amylosucrase is Neisseria polysaccharea amylosucrase. Optionally, the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 2. Optionally, the variant amylosucrase comprises or consists of an amino acid sequence having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Optionally, the 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 includes 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) includes at least two enzymes that can collectively or in combination convert sucrose to amylose. In some cases, the enzyme mixture includes sucrose phosphorylase. In some cases, sucrose phosphorylase can convert sucrose to glucose-1-phosphate. In some cases, contacting (a) further includes 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 includes, or consists of, an amino acid sequence of any one of SEQ ID NOs: 17-20, or an amino acid sequence having at least about 70% sequence identity with any one of the amino acid sequences of SEQ ID NOs: 17-20. In some cases, the enzyme mixture includes alpha-glucan phosphorylase. In some cases, alpha-glucan phosphorylase can convert glucose-1-phosphate to amylose. In some cases, contacting (a) further includes 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 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 an enzyme capable of producing a higher amount and / or concentration of gamma - cyclodextrin compared to alpha - cyclodextrin, beta - cyclodextrin, or both. In some cases, the enzyme capable of converting amylose to cyclodextrin in (b) is cyclodextrin glucanotransferase. In some cases, the cyclodextrin glucanotransferase is Bacillus clarkii cyclodextrin glucanotransferase. In some cases, the cyclodextrin glucanotransferase comprises, or consists of, the amino acid sequence of SEQ ID NO: 25 or 26, or an amino acid sequence having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO: 25 or 26. In some cases, the enzyme capable of converting amylose to cyclodextrin is variant cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase is capable of producing a higher amount and / or concentration of gamma - cyclodextrin compared to alpha - cyclodextrin, beta - cyclodextrin, or both, relative to wild - type cyclodextrin glucanotransferase. In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild - type cyclodextrin glucanotransferase.In some cases, the wild-type cyclodextrin glucanotransferase is the Bacillus clarkii cyclodextrin glucanotransferase. In some cases, the wild-type cyclodextrin glucanotransferase comprises or consists of the amino acid sequence of SEQ ID NO: 25 or 26. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO: 25 or 26. In some cases, at least one amino acid variant comprises at least one amino acid substitution. In some cases, at least one amino acid substitution comprises an amino acid substitution at position 186 of the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26. In some cases, the amino acid substitution at position 186 is Y186W. In some cases, at least one amino acid substitution comprises an amino acid substitution at position 223 of the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26. In some cases, the amino acid substitution at position 223 is selected from the group consisting of A223H, A223K, and A223R. In some cases, the contacting of (a) and the contacting of (b) are performed sequentially. In some cases, the contacting of (a) and the contacting of (b) are performed simultaneously or substantially simultaneously. In some cases, the amylose produced in (a) is not purified or isolated before the contacting of (b). In some cases, the contacting of (a), the contacting of (b), or both are performed in vitro. In some cases, the contacting of (a), the contacting of (b), or both are performed in a container, vial, bottle, test tube, well, plate, or enclosure. In some cases, at least one enzyme of the enzyme or enzyme mixture of (a), the variant enzyme of (b), or both are purified enzymes, isolated enzymes, or both. In some cases, at least one enzyme of the enzyme or enzyme mixture of (a), the variant enzyme of (b), or both are recombinantly produced enzymes.In some cases, contacting (a), contacting (b), or both are performed in vivo. In some cases, contacting (a), contacting (b), or both are performed in a recombinant host cell. In some cases, the recombinant host cell contains a heterologous nucleic acid encoding at least one enzyme of the enzyme or enzyme mixture of (a), the variant enzyme of (b), or both. In some cases, the recombinant host cell is a microbial cell. In some cases, the microbial cell is a bacterial cell. In some cases, 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 gamma-cyclodextrin to alpha-cyclodextrin in the composition containing cyclodextrin is at least 2:1. In some cases, the ratio of gamma-cyclodextrin to beta-cyclodextrin in the composition containing cyclodextrin is at least 2:1. In some cases, the composition containing cyclodextrin does not contain or substantially does not contain alpha-cyclodextrin, beta-cyclodextrin, or both.

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

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

Brief Description of the Drawings

[0006]

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Mode for Carrying Out the Invention

[0007] Current cyclodextrin production methods have problems such as supply chain shortages and issues related to scalability, quality variability, purification, and the cost of goods. Furthermore, there are several major drawbacks regarding 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 method of the present disclosure overcomes these problems by providing a method for the facile enzymatic synthesis of cyclodextrin-containing compositions from sucrose as a starting material, preferably a method for one-pot enzymatic synthesis.

[0008] The present specification provides methods for producing compositions containing cyclodextrin. The present specification also provides methods for the enzymatic synthesis of gamma-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. The present specification also provides methods for the enzymatic conversion of sucrose to gamma-cyclodextrin using various enzymes. In various aspects, the methods provided herein generally result in a higher production of gamma-cyclodextrin than alpha-cyclodextrin, beta-cyclodextrin, or both. In some cases, the methods provided herein produce gamma-cyclodextrin at a higher ratio than alpha-cyclodextrin, gamma-cyclodextrin, or both. The method generally includes the enzymatic conversion of sucrose to amylose as the first step (step (a)) of the synthetic pathway. In one embodiment, the method involves the use of a single enzyme (e.g., amylosucrase) to convert sucrose to amylose. In another embodiment, the method involves the use of two enzymes (e.g., sucrose phosphorylase and alpha-glucan phosphorylase) to convert sucrose to amylose. The method also generally includes the enzymatic conversion of amylose to gamma-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, purified and / or isolated (e.g., recombinant) enzymes).

[0009] 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. Natural cyclodextrin (CD) can be ingested without being significantly absorbed, so it has received "safe food certification" from the FDA and is generally regarded as a molecule with "GRAS status".

[0010] Gamma-cyclodextrin is widely used in the pharmaceutical industry. Various derivatives of gamma-cyclodextrin have been produced to improve the oral bioavailability and solubility of cyclodextrin. For example, modifying the hydroxyl groups of cyclodextrin with alkylhydroxyl groups dramatically improves the solubility of cyclodextrin. Some of the possible derivatives include gamma-cyclodextrin in which all eight primary hydroxyl groups are replaced with 2-(carboxyethyl)sulfanyl groups, or randomly methylated gamma-cyclodextrin and branched gamma-cyclodextrin.

[0011] In one aspect of the present disclosure, a method for producing a composition containing cyclodextrin is provided. Optionally, the method includes (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 includes (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 containing cyclodextrin. Optionally, the enzyme capable of converting amylose to cyclodextrin is an enzyme capable of producing gamma-cyclodextrin in an amount and / or concentration (e.g., weight %, mol %, or w / v) higher than that of alpha-cyclodextrin, beta-cyclodextrin, or both. Optionally, the composition containing cyclodextrin contains gamma-cyclodextrin and may further contain, as appropriate, beta-cyclodextrin, alpha-cyclodextrin, or any combination thereof. Optionally, the composition containing cyclodextrin contains gamma-cyclodextrin in an amount and / or concentration (e.g., weight %, mol %, or w / v) higher than that of alpha-cyclodextrin, beta-cyclodextrin, or both. Optionally, the amounts and / or concentrations of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin are measured by high performance liquid chromatography (HPLC).

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

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

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

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

[0016] In some cases, at least one variant contains at least one amino acid substitution relative to wild-type amylosucrase. In some cases, at least one amino acid variant contains at least one amino acid substitution relative to wild-type Cellulomonas carboniz T26 amylosucrase. In some cases, at least one amino acid variant contains at least one amino acid substitution relative to wild-type Neisseria polysaccharea amylosucrase. In some cases, at least one amino acid substitution includes 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 replaced by 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 (for example, 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 (for example, 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 (for example, 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 (for example, 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 (for example, 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 (for example, 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 (for example, 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 (for example, 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 (for example, SEQ ID NO: 16 in Table 2).

[0017] In some embodiments, the variant amylosucrase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 3 to 16 or 31 shown in Table 2, or 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 90%, 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 an amino acid sequence according to any one of SEQ ID NOs: 3 to 16 or 31 shown in Table 2. In a preferred embodiment, the variant amylosucrase comprises, or consists of, an amino acid sequence according to any one of SEQ ID NOs: 3 to 9 shown in Table 2. In some cases, the variant amylosucrase 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), preferably at least about 90% sequence identity, when compared to an amino acid sequence of any one of SEQ ID NOs: 3 to 16 shown in Table 2. In a preferred embodiment, the variant amylosucrase comprises, or consists of, an amino acid sequence 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), preferably at least about 90% sequence identity, when compared to an amino acid sequence of any one of SEQ ID NOs: 3 to 9 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 the specified amino acid substitution (i.e., the sequence identity is calculated based on the entire amino acid sequence of the variant enzyme including the specified amino acid substitution). Optionally, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 234 relative to SEQ ID NO:2 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, R234K, R234I, R234D, R234Y, R234W, R234E, R234L, and R234H.In a preferred embodiment, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 234 of SEQ ID NO: 2 selected from the group consisting of R234Q, R234G, R234A, R234S, R234M, R234C, and R234K. Optionally, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234Q of SEQ ID NO: 2. Optionally, the variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 2, preferably at least about 90% sequence identity, and an amino acid substitution R234G of SEQ ID NO: 2.In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234A relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234S relative to SEQ ID NO:2. In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and the amino acid substitution R234M relative to SEQ ID NO:2.In some cases, variant amylosucrase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO:2, preferably at least about 90% sequence identity, and 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 R. 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 includes contacting sucrose with an enzyme mixture capable of converting sucrose to amylose under conditions that allow for the conversion of sucrose to amylose, thereby producing amylose. Optionally, the method includes contacting sucrose with an enzyme mixture that includes at least two enzymes that can collectively or combinatorially convert sucrose to amylose. For example, the enzyme mixture can include at least sucrose phosphorylase and alpha-glucan phosphorylase. The method can include contacting sucrose with at least two enzymes simultaneously or substantially simultaneously. Alternatively, the method can include contacting sucrose with at least two enzymes sequentially. Figure 2B shows a schematic of a two-enzyme method for producing amylose from sucrose. In this example, sucrose is contacted with sucrose phosphorylase to convert sucrose to glucose-1-phosphate. Next, glucose-1-phosphate is contacted with alpha-glucan phosphorylase to convert glucose-1-phosphate to amylose. Optionally, sucrose phosphorylase and alpha-glucan phosphorylase are contacted with sucrose simultaneously or substantially simultaneously. In other cases, sucrose phosphorylase and alpha-glucan phosphorylase are added sequentially (e.g., sucrose phosphorylase is first contacted with sucrose to produce glucose-1-phosphate, and then alpha-glucan phosphorylase is added to produce amylose). Optionally, glucose-1-phosphate generated from the reaction with sucrose phosphorylase is isolated and / or purified before contacting it with alpha-glucan phosphorylase. In other cases, glucose-1-phosphate generated from the reaction with sucrose phosphorylase is not isolated and / or purified before contacting it with alpha-glucan phosphorylase. As used in the context of adding two or more components to the reaction mixture described herein, the term "substantially simultaneously" means that the two or more components are added to the reaction mixture within 10 seconds of each other.

[0021] In some cases, the sucrose phosphorylase is a wild-type sucrose phosphorylase. For example, the wild-type sucrose phosphorylase can be Bifidobacterium longum sucrose phosphorylase (e.g., NCBI accession number AAO84039). In some cases, the wild-type Bifidobacterium longum sucrose phosphorylase can contain or consist of 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 contain or consist of 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 contain or consist of 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 at one or more 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 position 140 for SEQ ID NO: 19 is K140M. In some cases, the amino acid substitution at position 144 for SEQ ID NO: 19 is Q144R. In some cases, the amino acid substitution at position 155 for SEQ ID NO: 19 is N155S. In some cases, the amino acid substitution at position 249 for SEQ ID NO: 19 is D249G. In some cases, the variant sucrose phosphorylase has amino acid substitutions T47S, S62P, Y77H, V128L, K140M, Q144R, N155S, and D249G relative to SEQ ID NO: 19. In some cases, the variant sucrose phosphorylase enzyme comprises or consists of the amino acid sequence according to SEQ ID NO: 20. Table 3 below shows non-limiting examples of sucrose phosphorylase enzymes (and their amino acid sequences) that can be used according to the methods provided herein.

Table 3-1

Table 3-2

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

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

[0024] In some embodiments, the alpha - glucan phosphorylase is a wild - type alpha - glucan phosphorylase. In some cases, the wild - type alpha - glucan phosphorylase can be a Solanum tuberosum alpha - glucan phosphorylase (e.g., NCBI accession number D00520.1). In some cases, the wild - type Solanum tuberosum alpha - glucan phosphorylase can contain or consist of 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 contain or consist of 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 contain or consist of 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: 21, preferably at least about 90% sequence identity, and includes the amino acid substitutions F39L, N135S, and T706I relative to SEQ ID NO: 21.

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

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

[0028] In some embodiments, the enzyme capable of converting amylose to cyclodextrin includes cyclodextrin glucanotransferase. Figure 3 shows the enzymatic conversion of amylose to gamma-cyclodextrin using cyclodextrin glucanotransferase. Preferably, the gamma-cyclodextrin glucanotransferase produces gamma-cyclodextrin from amylose in an amount and / or concentration higher than the amount and / or concentration of alpha-cyclodextrin and / or beta-cyclodextrin. In some cases, the cyclodextrin glucanotransferase is a wild-type cyclodextrin glucanotransferase. In some cases, the wild-type cyclodextrin glucanotransferase is Bacillus clarkii cyclodextrin glucanotransferase (e.g., NCBI accession number AB432985.1). In some cases, the wild-type cyclodextrin glucanotransferase comprises, consists of, or has an amino acid sequence with at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) to the amino acid sequence of SEQ ID NO: 25 or 26, preferably at least about 90% sequence identity.

[0029] In some cases, the cyclodextrin glucanotransferase is 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. The variant cyclodextrin glucanotransferase may comprise 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 is capable of producing cyclodextrin in a higher amount and / or concentration from amylose compared to alpha-cyclodextrin and / or beta-cyclodextrin relative to the wild-type cyclodextrin glucanotransferase.

[0030] In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type Bacillus clarkii cyclodextrin glucanotransferase (e.g., NCBI accession number AB432985.1; e.g., SEQ ID NO: 25 or 26). In some cases, the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to SEQ ID NO: 25 or SEQ ID NO: 26. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of wild-type Bacillus clarkii cyclodextrin glucanotransferase, preferably at least about 90% sequence identity. In some cases, the variant cyclodextrin glucanotransferase comprises or consists of an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 25 or SEQ ID NO: 26, preferably at least about 90% sequence identity.

[0031] In some cases, at least one amino acid variant contains at least one amino acid substitution relative to wild-type cyclodextrin glucanotransferase. In some cases, at least one amino acid substitution includes an amino acid substitution at position 186 relative to the amino acid sequence of SEQ ID NO: 26. In some cases, the amino acid substitution at position 186 relative to the amino acid sequence of SEQ ID NO: 26 is Y186W (e.g., SEQ ID NO: 27 in Table 5). In some cases, at least one amino acid substitution includes an amino acid substitution at position 223 relative to the amino acid sequence of SEQ ID NO: 26. In some cases, the amino acid substitution at position 223 relative to the amino acid sequence of SEQ ID NO: 26 is A223H (e.g., SEQ ID NO: 28 in Table 5). In some cases, the amino acid substitution at position 223 relative to the amino acid sequence of SEQ ID NO: 26 is A223K (e.g., SEQ ID NO: 29 in Table 5). In some cases, the amino acid substitution at position 223 relative to the amino acid sequence of SEQ ID NO: 26 is A223R (e.g., SEQ ID NO: 30 in Table 5).

[0032] 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. In some embodiments, the 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), preferably at least about 90% sequence identity, to any one of the amino acid sequences of SEQ ID NOs: 25 - 30 shown in Table 5.

Table 5-1

Table 5-2

Table 5-3

Table 5-4

[0033] 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, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 186 relative to SEQ ID NO: 26. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26, preferably at least about 90% sequence identity, and an amino acid substitution Y186W relative to SEQ ID NO: 26.

[0034] 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, preferably at least about 90% sequence identity, and an amino acid substitution at amino acid position 223 relative to SEQ ID NO: 26. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26, preferably at least about 90% sequence identity, and the amino acid substitution A223H relative to SEQ ID NO: 26. Optionally, the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity (e.g., at least about 75%, at least about 80%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more) with the amino acid sequence of SEQ ID NO: 26, preferably at least about 90% sequence identity, and the amino acid substitution A223K relative to SEQ ID NO: 26.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, preferably at least about 90% sequence identity, and the amino acid substitution A223R with respect to SEQ ID NO: 26.

[0035] In some embodiments, the cyclodextrin glucanotransferase is derived from a microbial cell. In some cases, the cyclodextrin glucanotransferase 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 cyclodextrin glucanotransferase is derived from Bacillus clarkii. In some embodiments, the cyclodextrin glucanotransferase can be produced within a microbial cell. In some embodiments, the cyclodextrin glucanotransferase is expressed within a recombinant host cell (e.g., from a recombinant polynucleotide). In some cases, the cyclodextrin glucanotransferase is produced recombinantly. In some cases, the cyclodextrin glucanotransferase is produced in a yeast cell (e.g., produced recombinantly). In some cases, the yeast cell is a Pichia yeast cell such as a Pichia pastoris cell.

[0036] In various embodiments, the methods provided herein produce gamma-cyclodextrin in a higher ratio than alpha-cyclodextrin, beta-cyclodextrin, or both. For example, in some cases, the methods provided herein provide a ratio of gamma-cyclodextrin to alpha-cyclodextrin, beta-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 gamma-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 gamma-cyclodextrin to beta-cyclodextrin of at least 3:1. 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. In a preferred embodiment, the methods provided herein provide a ratio of beta-cyclodextrin to both alpha-cyclodextrin and gamma-cyclodextrin of at least 3:1. 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.

[0037] Throughout the present disclosure, methods for achieving robust enzyme activity at each step are outlined to obtain gamma-cyclodextrin in a higher yield than is currently achievable. 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 gamma-cyclodextrin (e.g., as described herein) is carried out over a second period, thereby enabling a catalytic conversion of amylose to gamma-cyclodextrin. In some embodiments, the first enzymatic reaction (e.g., converting sucrose to amylose, as described herein, for example) and the second enzymatic reaction (e.g., converting amylose to gamma-cyclodextrin, as described herein, for example) are carried out in the same reservoir (e.g., one-pot synthesis method).

[0038] 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 added again after a certain period has elapsed to promote catalytic activity. In some embodiments, sucrose is added once at the start of the reaction period and then added again after a certain period 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 gamma-cyclodextrin).In some embodiments, the enzyme used in the first enzyme reaction step (e.g., as described herein, e.g., to convert sucrose to amylose) is added at a different time (e.g., prior thereto) than the enzyme used in the second enzyme reaction step (e.g., to convert amylose to gamma-cyclodextrin).

[0039] 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.

[0040] In some embodiments, the reaction time is an important consideration for obtaining gamma-cyclodextrin in maximum yield. In some embodiments, the production of gamma-cyclodextrin can involve the decomposition of the product into glucose, maltose, and other sugars. Therefore, it is important to obtain gamma-cyclodextrin without decomposing it. In some embodiments, the entire reaction (e.g., method step (a) and method step (b)) is carried out over 12 hours or less. In some embodiments, the entire reaction (e.g., method step (a) and method step (b)) is carried out over 8 hours or less. In some embodiments, the entire reaction is carried out over 7 hours or less. In some embodiments, the entire reaction is carried out over 6 hours or less. In some embodiments, the entire reaction is carried out over 5 hours or less. In some embodiments, the entire reaction is carried out over 4 hours or less. In some embodiments, the entire reaction is carried out over 3 hours or less. In some embodiments, the entire reaction is carried out over 2 hours or less. In some embodiments, the entire reaction is carried out over 1 hour or less.

[0041] Temperature is an important consideration for maximizing the yield of gamma-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, about 50°C, or about 55°C.

[0042] 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 approximately the same temperature. For example, the enzymatic reaction of step (a) is preferably carried out at about 50°C or about 55°C. In this embodiment, it is preferred that the enzymatic reaction of step (b) is also carried out at about 50°C or about 55°C, respectively.

[0043] In one-pot synthesis, even though the optimal temperature of each enzyme may vary slightly, it is considered that the functionality of the enzyme mixture(s) should be maximized.

[0044] In some embodiments, the reaction is carried out at a pH of 5.0 to 7.5, such as 6.0 to 7.0, such as about 7.0. In some embodiments, the reaction is carried out at pH 6.0. In some embodiments, the reaction is carried out at pH 7.0.

[0045] 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 mixture at a concentration of about 50 mM to about 200 mM. In a preferred embodiment, the buffer is present at a concentration of about 100 mM.

[0046] In some embodiments, the reaction is carried out in a reservoir having a reservoir volume of about 1 mL to about 1,000,000 L. For example, the reaction may be carried out in a reservoir having a reservoir volume of about 100 mL to about 10 L, such as a reservoir volume of about 500 mL or about 10 L.

[0047] 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.

[0048] In some embodiments, the reaction is carried out in a stirred tank reactor (STR), a loop reactor, a plug flow reactor, a single-stage or multi-stage continuous stirred tank reactor, or any other suitable reactor known in the art. In some embodiments, the reaction is carried out in a stirred tank reactor and the reactants are stirred at about 100 to about 200 rpm, such as about 160 rpm.

[0049] In some embodiments, one or more of the enzymatic reactions are carried out in a reaction mixture comprising 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 the organic solvent surprisingly increases the yield of gamma-cyclodextrin obtained from the enzymatic reaction. For example, the addition of the organic solvent can increase the yield of gamma-cyclodextrin by at least about 5%, such as at least about 10%, such as at least about 15%, such as at least about 20%, such as at least about 50%, such as at least about 100%, such as at least about 150%, such as at least about 200%, such as at least about 250%, such as at least about 300%, such as at least about 350%, such as at least about 400% compared to the yield obtained from the enzymatic reaction carried out without the organic solvent. It is believed that when the organic solvent is added, the solubility of gamma-cyclodextrin in the reaction mixture decreases, causing gamma-cyclodextrin to precipitate, and the concentration of gamma-cyclodextrin in the reaction mixture decreases, thereby increasing the yield of gamma-cyclodextrin. This prevents the degradation of gamma-cyclodextrin by the enzyme.

[0050] In some embodiments, the amount of the organic solvent (preferably toluene) in the reaction mixture is from about 0.1 v / v% to about 40 v / v% of the reaction mixture, such as from about 1 v / v% to about 35 v / v%, such as from about 5 v / v% to about 25 v / v%.

[0051] 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).

[0052] 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.

[0053] 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.

[0054] In some embodiments, the enzyme is provided in the 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.

[0055] In certain embodiments, any one of the enzyme reactions provided herein (e.g., the first enzyme reaction of converting sucrose to amylose and / or the second enzyme reaction of converting amylose to gamma - cyclodextrin) can occur within a microbial host cell. 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 may be fed to the microbial host cell, and the conversion from sucrose to gamma - cyclodextrin may occur within the microbial host cell.

[0056] 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) may be a variant amylosucrase (e.g., this variant amylosucrase may 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) may be a variant cyclodextrin glucanotransferase, and the enzyme may be immobilized on a resin. Optionally, the enzyme or enzyme mixture of (a) and the enzyme of (b) are immobilized on the same resin.

[0057] The resin-immobilized enzyme can be reused by the methods described herein. However, the inventors have found that when the resin-immobilized enzyme is reused, the yield of gamma-cyclodextrin tends to decrease. This is thought to be due to the enzyme leaching from the resin during use, resulting in a decrease in the enzyme conversion rate. Therefore, it is desirable to improve the enzyme stability on the resin and thereby prevent enzyme leaching. This is because it enables the resin-immobilized enzyme to be reused more frequently and / or at a higher enzyme conversion rate, thereby increasing the yield of the reaction.

[0058] The inventors have found that enzyme stability can be improved by using freeze-dried enzymes, spray-drying the enzymes, and / or introducing additives.

[0059] 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 in a buffer (such as sodium citrate buffer), lysed, and centrifuged 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.

[0060] 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%, such as about 0.5 w / v% to about 5 w / v% of the cell slurry or whole cell lysate. 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 a preferred embodiment, the additive is mannitol, sorbitol, sucrose, or a combination thereof.

[0061] In some embodiments, the cell slurry or cell lysate may be lyophilized. For example, the cell slurry or cell lysate may be lyophilized over a period of two days. Methods of lyophilization are known in the art.

[0062] The inventors have found that adding an additive to a cell slurry or whole cell lysate (as described above) increases enzyme stability compared to a cell slurry or whole cell lysate that does not contain the additive, and that lyophilizing a cell slurry or whole cell lysate (as described above) increases enzyme stability compared to a 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.

[0063] In some embodiments, the methods described herein produce a composition comprising at least 2 g / L of gamma-cyclodextrin. In some embodiments, the method produces a composition comprising at least 3 g / L of gamma-cyclodextrin, at least 4 g / L of gamma-cyclodextrin, at least 5 g / L of gamma-cyclodextrin, at least 6 g / L of gamma-cyclodextrin, at least 7 g / L of gamma-cyclodextrin, at least 8 g / L of gamma-cyclodextrin, at least 9 g / L of gamma-cyclodextrin, at least 10 g / L of gamma-cyclodextrin, at least 12 g / L of gamma-cyclodextrin, at least 15 g / L of gamma-cyclodextrin, at least 20 g / L of gamma-cyclodextrin, at least 30 g / L of gamma-cyclodextrin, at least 40 g / L of gamma-cyclodextrin, at least 50 g / L of gamma-cyclodextrin, or at least 60 g / L of gamma-cyclodextrin. In a preferred embodiment, the method produces a composition comprising at least 10 g / L of gamma-cyclodextrin.

[0064] In some embodiments, the yield percentage of gamma-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 gamma-cyclodextrin produced by the methods described herein by the maximum theoretical amount of gamma-cyclodextrin that could be produced from the sucrose reagent of the starting material.

[0065] Also provided herein is a composition comprising cyclodextrin, said cyclodextrin comprising gamma-cyclodextrin and optionally further comprising alpha-cyclodextrin, beta-cyclodextrin, or any combination thereof, and said composition comprising cyclodextrin comprises gamma-cyclodextrin in an amount and / or concentration higher than alpha-cyclodextrin, beta-cyclodextrin, or both. Preferably, the composition is obtained from the method provided herein. The composition may comprise a ratio of gamma-cyclodextrin to alpha-cyclodextrin, beta-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.

[0066] 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 gamma-cyclodextrin than a wild-type enzyme capable of converting amylose to cyclodextrin, the composition comprising cyclodextrin comprises gamma-cyclodextrin and may further comprise alpha-cyclodextrin, beta-cyclodextrin, or any combination thereof, the ratio of gamma-cyclodextrin in the composition to alpha-cyclodextrin, beta-cyclodextrin, or both is at least 5:1, 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.

[0067] Also provided herein is gamma-cyclodextrin. Preferably, the gamma-cyclodextrin is obtained from the method provided herein.

[0068] Also provided herein is the use of sucrose as a starting material for the production of gamma-cyclodextrin. Also provided herein is the use of sucrose in a method for producing gamma-cyclodextrin, the method not using starch.

[0069] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for converting sucrose to amylose.

[0070] Also provided herein is the use of any one of the enzymes described herein for converting amylose to cyclodextrin and / or for producing gamma-cyclodextrin in an amount and / or concentration higher than that of alpha-cyclodextrin, beta-cyclodextrin, or both.

[0071] Also provided herein is the use of any one of the enzymes or enzyme mixtures described herein for the production of gamma-cyclodextrin, which production does not require starch as a starting material.

[0072] 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 to 31. 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 to 31.

[0073] Preferably, the enzyme is a variant amylosucrase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 3 to 16 or 31. 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 to 16 or 31.

[0074] 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, to the amino acid sequence of SEQ ID NO: 20.

[0075] 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, to the amino acid sequence of SEQ ID NO: 24.

[0076] Preferably, this enzyme is a variant cyclodextrin glucanotransferase enzyme comprising or consisting of any one of the amino acid sequences of SEQ ID NOs: 27 to 30. 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, to any one of the amino acid sequences of SEQ ID NOs: 27 to 30.

[0077] Also provided herein are enzyme compositions comprising one or more of the enzymes described herein.

[0078] 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.

[0079] Also provided herein is the use of an organic solvent, preferably toluene, to increase the yield of gamma-cyclodextrin obtained in a method for producing gamma-cyclodextrin, such as gamma-cyclodextrin obtained from any one of the methods described herein.

[0080] 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 sophisticated BLAST computer programs available from the National Institutes of Health (including version 2.2.9). 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 in the blastp program. In this program, a SEG filter can also be used to mask segments of the query sequence determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17:149-163 (1993).The desired range of sequence identity is approximately 70% to 100%, and integer values in between. Generally, 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.

[0081] As used herein, the term "about" generally refers to a range that is 15% more or less than the numerical value described within the context of a particular usage. For example, "about 10" includes the range from 8.5 to 11.5.

[0082] 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".

[0083] As used herein, "a", "an", and "the" can include plural referents unless explicitly and specifically limited to one referent. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0084] 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 following number, regardless of the order in which they are described.

[0085] 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 composition containing cyclodextrin contains gamma-cyclodextrin and may further contain, optionally, alpha-cyclodextrin, beta-cyclodextrin, or any combination thereof, and wherein the composition containing cyclodextrin contains gamma-cyclodextrin in an amount and / or concentration higher than that of alpha-cyclodextrin, beta-cyclodextrin, or both.

[0086] Embodiment 2: The method according to Embodiment 1, wherein the enzyme in (a) is amylosucrase or the enzyme mixture in (a) contains amylosucrase.

[0087] 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.

[0088] Embodiment 4: The method according to Embodiment 3, wherein the variant amylosucrase is capable of producing a higher amount and / or concentration of amylose from sucrose relative to wild-type amylosucrase.

[0089] Embodiment 5: The method according to Embodiment 3 or 4, wherein the wild-type amylosucrase is Cellulomonas carboniz T26 amylosucrase.

[0090] Embodiment 6: The method according to embodiment 5, wherein the wild-type amylosucrase comprises or consists of the amino acid sequence of SEQ ID NO: 1.

[0091] Embodiment 7: The method according to embodiment 3 or 4, wherein the wild-type amylosucrase is Neisseria polysaccharea amylosucrase.

[0092] 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.

[0093] 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 with the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

[0094] 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 relative to the wild-type amylosucrase.

[0095] Embodiment 11: The method according to embodiment 10, wherein the at least one amino acid substitution comprises an amino acid substitution at position 234 relative to the wild-type amylosucrase having the amino acid sequence of SEQ ID NO: 2.

[0096] 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.

[0097] Embodiment 13: The method according to embodiment 1, wherein the enzyme mixture of (a) comprises at least two enzymes that can collectively or in combination convert sucrose to amylose.

[0098] Embodiment 14: The method according to embodiment 13, wherein the enzyme mixture contains sucrose phosphorylase.

[0099] Embodiment 15: The method according to embodiment 14, wherein the sucrose phosphorylase can convert sucrose into glucose-1-phosphate.

[0100] Embodiment 16: The method according to embodiment 15, further comprising contacting the sucrose with the sucrose phosphorylase under conditions that enable the conversion of the sucrose into glucose-1-phosphate in (a).

[0101] 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.

[0102] Embodiment 18: The method according to any one of embodiments 14 to 17, wherein the sucrose phosphorylase contains, or consists of, an amino acid sequence of any one of SEQ ID NOs: 17 to 20, or an amino acid sequence having at least about 70% sequence identity with an amino acid sequence of any one of SEQ ID NOs: 17 to 20.

[0103] Embodiment 19: The method according to one of embodiments 13 to 18, wherein the enzyme mixture contains alpha-glucan phosphorylase.

[0104] Embodiment 20: The method according to embodiment 19, wherein the alpha-glucan phosphorylase can convert the glucose-1-phosphate into amylose.

[0105] Embodiment 21: The method according to embodiment 20, wherein the contacting in (a) further comprises contacting the glucose-1-phosphate with the alpha-glucan phosphorylase under conditions that allow conversion of the glucose-1-phosphate to amylose.

[0106] 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.

[0107] 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 with any one of the amino acid sequences of SEQ ID NOs: 21 to 24.

[0108] Embodiment 24: The method according to any one of embodiments 1 to 23, wherein the enzyme capable of converting the amylose in (b) to cyclodextrin comprises an enzyme capable of producing a higher amount and / or concentration of gamma-cyclodextrin compared to alpha-cyclodextrin, beta-cyclodextrin, or both.

[0109] Embodiment 25: The method according to any one of embodiments 1 to 24, wherein the enzyme capable of converting the amylose in (b) to cyclodextrin is cyclodextrin glucanotransferase.

[0110] Embodiment 26: The method according to embodiment 25, wherein the cyclodextrin glucanotransferase is Bacillus clarkii cyclodextrin glucanotransferase.

[0111] Embodiment 27: The method according to Embodiment 25 or 26, wherein the cyclodextrin glucanotransferase comprises, or consists of, the amino acid sequence of SEQ ID NO: 25 or 26, or an amino acid sequence having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO: 25 or 26.

[0112] Embodiment 28: The method according to any one of Embodiments 1 to 27, wherein the enzyme capable of converting amylose into cyclodextrin is a variant cyclodextrin glucanotransferase.

[0113] Embodiment 29: The method according to Embodiment 28, wherein the variant cyclodextrin glucanotransferase is capable of producing alpha-cyclodextrin, beta-cyclodextrin, or a higher amount and / or concentration of gamma-cyclodextrin than both, relative to wild-type cyclodextrin glucanotransferase.

[0114] Embodiment 30: The method according to Embodiment 29, wherein the variant cyclodextrin glucanotransferase comprises at least one amino acid variant relative to wild-type cyclodextrin glucanotransferase.

[0115] Embodiment 31: The method according to Embodiment 30, wherein the wild-type cyclodextrin glucanotransferase is Bacillus clarkii cyclodextrin glucanotransferase.

[0116] Embodiment 32: The method according to any one of Embodiments 29 to 31, wherein the wild-type cyclodextrin glucanotransferase comprises, or consists of, the amino acid sequence of SEQ ID NO: 25 or 26.

[0117] Embodiment 33: The method according to any one of Embodiments 28 to 32, wherein the variant cyclodextrin glucanotransferase comprises, or consists of, an amino acid sequence having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO: 25 or 26.

[0118] Embodiment 34: The method according to any one of Embodiments 30 to 33, wherein the at least one amino acid variant comprises at least one amino acid substitution.

[0119] Embodiment 35: The method according to Embodiment 34, wherein the at least one amino acid substitution comprises an amino acid substitution at position 186 of the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26.

[0120] Embodiment 36: The method according to Embodiment 35, wherein the amino acid substitution at position 186 is Y186W.

[0121] Embodiment 37: The method according to any one of Embodiments 34 to 36, wherein the at least one amino acid substitution comprises an amino acid substitution at position 223 of the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26.

[0122] Embodiment 38: The method according to Embodiment 37, wherein the amino acid substitution at position 223 is selected from the group consisting of A223H, A223K, and A223R.

[0123] Embodiment 39: The method according to any one of Embodiments 1 to 38, wherein the contacting in (a) and the contacting in (b) are performed sequentially.

[0124] Embodiment 40: The method according to any one of Embodiments 1 to 38, wherein the contacting in (a) and the contacting in (b) are performed simultaneously or substantially simultaneously.

[0125] The method according to any one of Embodiments 1 to 40, wherein the amylose produced in (a) is not purified or isolated before the contacting in (b).

[0126] The method according to any one of Embodiments 1 to 41, wherein the contacting in (a), the contacting in (b), or both are performed in vitro.

[0127] The method according to Embodiment 42, wherein the contacting in (a), the contacting in (b), or both are performed in a container, vial, bottle, test tube, well, plate, or enclosure.

[0128] The method according to Embodiment 42 or 43, 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.

[0129] The method according to any one of Embodiments 42 to 44, 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.

[0130] The method according to any one of Embodiments 1 to 41, wherein the contacting in (a), the contacting in (b), or both are performed in vivo.

[0131] The method according to Embodiment 46, wherein the contacting in (a), the contacting in (b), or both are performed in a recombinant host cell.

[0132] The method according to Embodiment 47, 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.

[0133] Embodiment 49: The method according to embodiment 47 or 48, wherein the recombinant host cell is a microbial cell.

[0134] Embodiment 50: The method according to embodiment 49, wherein the microbial cell is a bacterial cell.

[0135] Embodiment 51: The method according to any one of embodiments 1 to 50, wherein the ratio of gamma-cyclodextrin to alpha-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

[0136] Embodiment 52: The method according to any one of embodiments 1 to 51, wherein the ratio of gamma-cyclodextrin to beta-cyclodextrin in the composition containing cyclodextrin is at least 2:1.

[0137] Embodiment 53: The method according to any one of embodiments 1 to 52, wherein the composition containing cyclodextrin does not contain or substantially does not contain alpha-cyclodextrin, beta-cyclodextrin, or both.

Examples

[0138] Example 1. Variant cyclodextrin glucanotransferase can increase the production of gamma-cyclodextrin with respect to alpha-cyclodextrin and beta-cyclodextrin. This example demonstrates that the mutant cyclodextrin glucanotransferase enzyme was able to increase the production of gamma-cyclodextrin from amylose with respect to either or both of alpha-cyclodextrin and beta-cyclodextrin. In this example, several different cyclodextrin glucanotransferase enzymes ("BKcgt[W]" having the amino acid sequence according to SEQ ID NO: 27; "BKcgt[H]" having the amino acid sequence according to SEQ ID NO: 28; "BKcgt[K]" having the amino acid sequence according to SEQ ID NO: 29; and "BKcgt[R]" having the amino acid sequence according to SEQ ID NO: 30) were expressed in Escherichia coli and then separated from the insoluble cell debris mixture by centrifugation. The cyclodextrin glucanotransferase enzyme was exposed to soluble starch (60 g / L) in 0.1 M sodium citrate buffer at pH 8.0 at 55 °C for 1 hour. The reaction was quenched using formic acid, and the amounts of alpha-cyclodextrin, beta-cyclodextrin, and gamma-cyclodextrin were measured by HPLC. Figure 4 and Table 6 below demonstrate that all of the cyclodextrin glucanotransferase enzymes tested (BKcgt[W], BKcgt[H], BKcgt[K], and BKcgt[R]) were able to produce a higher ratio of gamma-cyclodextrin to beta-cyclodextrin. Alpha-cyclodextrin was not detected in any of the reactions.

Table 6

[0139] Example 2. One-pot synthesis of gamma-cyclodextrin from sucrose In this example, a two-enzyme system was used to produce gamma-cyclodextrin from sucrose (i.e., method step (a) was a one-enzyme method (e.g., as described herein), and method step (b) was a one-enzyme method (e.g., as described herein)). Amylosucrase R234Q (having the amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (BKcgt[R] having the amino acid sequence according to SEQ ID NO: 30) were expressed in Escherichia coli and then separated from the insoluble cell debris mixture by centrifugation. Next, 200 μL of amylosucrase (SEQ ID NO: 3) and various amounts of cyclodextrin glucanotransferase (SEQ ID NO: 30; 30 μL, 50 μL, and 100 μL) were exposed to 250 g / L sucrose at 50 °C at different pHs (pH 6.0, pH 7.0, and pH 8.0) in 0.1 M sodium citrate buffer. As shown in Figure 5A, the levels of gamma-cyclodextrin were measured by HPLC after 3 hours. The reaction was further optimized for pH using smaller pH increments between pH 6.5 and pH 7.5. This showed that the reaction functioned best at pH 7.0, but could produce reasonable amounts of product at both pH 6.5 and pH 7.5. The results appear to be independent of the vessel size and, as shown in Figure 5B, function best at times longer than 1 hour and at a pH of approximately 7.0.

[0140] Figures 5A and 5B demonstrate that a one-pot synthetic reaction can produce gamma-cyclodextrin from sucrose under various pH conditions. These reactions rely on a two-enzyme system composed of amylosucrase R234Q (having the amino acid sequence according to SEQ ID NO: 3) and cyclodextrin glucanotransferase (BKcgt[R] having the amino acid sequence according to SEQ ID NO: 30), starting with sucrose.

[0141] Example 3. Freeze-drying of cell slurries and cell lysates of amylosucrase containing additives The lysates and whole cell slurries of amylosucrase (having the amino acid sequence according to SEQ ID NO: 3) were lyophilized together with various additives. More specifically, 0.5 w / v%, 1.0 w / v%, or 5.0 w / v% of PEG, maltose, sorbitol, sucrose, glucose, mannitol, lactose, milk powder, starch, or beta-cyclodextrin was added to 1 mL of the lysate or cell slurry. Next, the mixture was lyophilized over a period of 2 days.

[0142] 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.

[0143] 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 whole cell slurry respectively). The results are shown in FIGS. 6A and 6B respectively.

[0144] As shown in FIG. 6, the addition of additives to amylosucrase before lyophilization improves the stability of the enzyme. In particular, it was demonstrated that the addition of 5% sucrose, 0.5% mannitol, and 0.5% sorbitol improved the enzyme activity retention rate of the cell lysate and whole cell slurry of amylosucrase.

[0145] This embodiment is presented for the purpose of exemplifying various embodiments of the present invention and is not intended to limit the present invention in any way. This embodiment, 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.

[0146] Although the preferred embodiments of the present invention have been shown and described herein, 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. It should be understood that in practicing the present invention, 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 and their equivalents within the scope of these claims are 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) The process involves 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, The composition comprising cyclodextrin comprises gamma-cyclodextrin and may optionally further comprise alpha-cyclodextrin, beta-cyclodextrin, or any combination thereof. The method wherein the composition comprising cyclodextrin comprises gamma-cyclodextrin in an amount and / or concentration higher than that of alpha-cyclodextrin, beta-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 contains or consists of the amino acid sequence of SEQ ID NO: 1, or (ii) Neisseria polysaccharea amylosculase, for example, the wild-type amylosculase contains or consists of 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 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 capable of collectively or in combination converting sucrose to amylose. 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, A) 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 B) 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, Selectively, the alpha-glucan phosphorylase, i) 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 ii) The method according to claim 3, comprising or consisting of one 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 with one of SEQ ID NOs. 21 to 24.

5. The enzyme that can convert the amylose in (b) to cyclodextrin is A) An enzyme capable of producing a higher amount and / or concentration of gamma-cyclodextrin compared to alpha-cyclodextrin, beta-cyclodextrin, or both, B) Cyclodextrin glucanotransferase, which can be selected arbitrarily. The cyclodextrin glucanotransferase is Bacillus clarkii cyclodextrin glucanotransferase, Preferably, the cyclodextrin glucanotransferase contains or comprises the amino acid sequence of SEQ ID NO: 25 or 26, or an amino acid sequence having at least about 70% sequence identity, preferably at least 90% sequence identity, with the amino acid sequence of SEQ ID NO: 25 or 26. Preferably, the wild-type cyclodextrin glucanotransferase contains or consists of the amino acid sequence of SEQ ID NO: 25 or 26, and / or C) Contains variant cyclodextrin glucanotransferase, optionally, The variant cyclodextrin glucanotransferase can produce gamma-cyclodextrin in higher amounts and / or concentrations than wild-type cyclodextrin glucanotransferase, compared to alpha-cyclodextrin, beta-cyclodextrin, or both. Furthermore, optionally, the variant cyclodextrin lucanotransferase comprises at least one amino acid variant relative to the wild-type cyclodextrin lucanotransferase, Preferably, the wild-type cyclodextrin glucanotransferase is Bacillus clarkii cyclodextrin glucanotransferase. Preferably, the wild-type cyclodextrin glucanotransferase contains or comprises the amino acid sequence of SEQ ID NO: 25 or 26. Preferably, the variant cyclodextrin glucanotransferase contains, or comprises, an amino acid sequence having at least about 70% sequence identity with the amino acid sequence of SEQ ID NO: 25 or 26. Furthermore, optionally, the at least one amino acid variant comprises at least one amino acid substitution. Furthermore, optionally, the at least one amino acid substitution may be: A) The amino acid substitution at position 186 of the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26, Preferably, the amino acid substitution at position 186 is Y186W, and / or B) The amino acid substitution at position 223 of the wild-type cyclodextrin glucanotransferase having the amino acid sequence of SEQ ID NO: 26, Preferably, the method according to any one of claims 1 to 4, wherein the amino acid substitution at position 223 is selected from the group consisting of A223H, A223K, and A223R.

6. The method according to any one of claims 1 to 4, wherein the contact in (a) and the contact in (b) are performed sequentially, or the contact in (a) and the contact in (b) are performed simultaneously or substantially simultaneously.

7. The method according to any one of claims 1 to 4, wherein the amylose produced in (a) is not purified or isolated before contact in (b).

8. (a) the contact, (b) the contact, or both are performed in vitro. Optionally, A) 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 B) 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.

9. (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.

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

1.

11. The method according to any one of claims 1 to 4, wherein the composition comprising cyclodextrin does not contain or substantially contains alpha-cyclodextrin, beta-cyclodextrin, or both.

12. The method according to any one of claims 1 to 4, wherein at least one enzyme from (a) the enzyme or the enzyme mixture, (b) the variant enzyme, or both are produced in Pichia yeast cells.

13. The method according to any one of claims 1 to 4, wherein the contact in (a) and / or the contact in (b) are carried out in a reaction mixture containing an organic solvent, preferably toluene.

14. (a) at least one of the enzymes or the enzyme mixture, (b) the variant enzyme, or both, A) Immobilized on a resin, and / or B) Provided in cell slurry or 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 combinations thereof, according to any one of claims 1 to 4.

15. The method according to claim 1, wherein the ratio of gamma-cyclodextrin in the composition to alpha-cyclodextrin, beta-cyclodextrin, or both is at least 5:1, 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 an organic solvent (preferably toluene), and the entire reaction is carried out over a period of 8 hours or less.

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

17. The composition according to claim 16, wherein the ratio of gamma-cyclodextrin to alpha-cyclodextrin in the composition containing cyclodextrin is at least 2:1, preferably at least 100:

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

1.

18. The composition according to claim 16, wherein the yield of gamma-cyclodextrin is at least about 10%, preferably at least about 60%.

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

20. The use of one or more enzymes from SEQ ID NO: 1 to SEQ ID NO: 31 for the production of gamma cyclodextrin, Preferably, the use is such that the manufacturing method is the method according to any one of claims 1 to 4.

21. An enzyme comprising or consisting of one amino acid sequence from Sequence ID No. 1 to 31, or an enzyme comprising or consisting of an amino acid sequence having at least about 70% sequence identity, preferably at least about 90% sequence identity, with one amino acid sequence from Sequence ID No. 1 to 31.

22. The enzyme according to claim 21, (i) Variant amyloscase enzymes containing or consisting of one amino acid sequence from sequence numbers 3-16 or 31, (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 of the amino acid sequences of SEQ ID NOs. 27-30 The enzyme, as described above.

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